Ph20 polypeptide variants, formulations and uses thereof

ABSTRACT

Modified PH20 hyaluronidase polypeptides, including modified polypeptides that exhibit increased stability and/or increased activity, are provided. Also provided are compositions and formulations and uses thereof.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/327,568, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USESTHEREOF,” and filed May 21, 2021, to Ge Wei, H. Michael Shepard, QipingZhao and Robert James Connor, which is a continuation of U.S.application Ser. No. 16/912,590, now issued on Jul. 20, 2021, as U.S.Pat. No. 11,066,656, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF,” and filed Jun. 25, 2020, to Ge Wei, H. MichaelShepard, Qiping Zhao and Robert James Connor, which is a continuation ofU.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, asU.S. Pat. No. 10,865,400, entitled “PH20 POLYPEPTIDE VARIANTS,FORMULATIONS AND USES THEREOF,” and filed on Aug. 2, 2016, to Ge Wei, H.Michael Shepard, Qiping Zhao and Robert James Connor, which is adivisional of U.S. application Ser. No. 13/694,731, now issued on Sep.20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, whichclaims the benefit of priority to U.S. Provisional Application Nos.61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012,respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF.”

U.S. application Ser. No. 17/327,568 also is a continuation of U.S.application Ser. No. 16/824,572, now issued on Jun. 22, 2021, as U.S.Pat. No. 11,041,149, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF,” and filed Mar. 19, 2020, to Ge Wei, H. MichaelShepard, Qiping Zhao and Robert James Connor, which is a continuation ofU.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, asU.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is adivisional of U.S. application Ser. No. 13/694,731, now issued on Sep.20, 2016, as U.S. Pat. No. 9,447,401, filed on Dec. 28, 2012, whichclaims the benefit of priority to U.S. Provisional Application Nos.61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012,respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF.”

U.S. application Ser. No. 17/327,568 also a continuation of U.S.application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S.Pat. No. 10,865,400, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF,” and filed on Aug. 2, 2016, to Ge Wei, H. MichaelShepard, Qiping Zhao and Robert James Connor, which is a divisional ofU.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, asU.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims thebenefit of priority to U.S. Provisional Application Nos. 61/631,313 and61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, andeach entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USESTHEREOF.”

U.S. application Ser. No. 17/327,568 also is a continuation of U.S.application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S.Pat. No. 9,447,401, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF,” and filed on Dec. 28, 2012, to Ge Wei, H. MichaelShepard, Qiping Zhao and Robert James Connor, which claims the benefitof priority to U.S. Provisional Application Nos. 61/631,313 and61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, andeach entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USESTHEREOF.”

This application also is a divisional of U.S. application Ser. No.17/327,586, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USESTHEREOF,” and filed May 21, 2021, to Ge Wei, H. Michael Shepard, QipingZhao and Robert James Connor, which is a continuation of U.S.application Ser. No. 16/912,590, now issued on Jul. 20, 2021, as U.S.Pat. No. 11,066,656, and filed Jun. 25, 2020, which is a continuation ofU.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, asU.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is adivisional of U.S. application Ser. No. 13/694,731, now issued on Sep.20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, whichclaims the benefit of priority to U.S. Provisional Application Nos.61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012,respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF.”

U.S. application Ser. No. 17/327,586 also is a continuation of U.S.application Ser. No. 16/824,572, now issued on Jun. 22, 2021, as U.S.Pat. No. 11,041,149, and filed Mar. 19, 2020, which is a continuation ofU.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, asU.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is adivisional of U.S. application Ser. No. 13/694,731, now issued on Sep.20, 2016, as U.S. Pat. No. 9,447,401, filed on Dec. 28, 2012, whichclaims the benefit of priority to U.S. Provisional Application Nos.61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012,respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONSAND USES THEREOF.” U.S. application Ser. No. 17/327,586 also acontinuation of U.S. application Ser. No. 15/226,489, now issued on Dec.15, 2020, as U.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, whichis a divisional of U.S. application Ser. No. 13/694,731, now issued onSep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012,which claims the benefit of priority to U.S. Provisional ApplicationNos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1,2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS,FORMULATIONS AND USES THEREOF.”

U.S. application Ser. No. 17/327,586 also is a continuation of U.S.application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S.Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefitof priority to U.S. Provisional Application Nos. 61/631,313 and61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, andeach entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USESTHEREOF.”

This application also is a divisional of U.S. application Ser. No.16/912,590, now issued on Jul. 20, 2021, as U.S. Pat. No. 11,066,656,and filed Jun. 25, 2020, which is a continuation of U.S. applicationSer. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No.10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S.application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S.Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefitof priority to U.S. Provisional Application Nos. 61/631,313 and61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, andeach entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USESTHEREOF.”

U.S. application Ser. No. 16/912,590, now issued on Jul. 20, 2021, asU.S. Pat. No. 11,066,656, filed Jun. 25, 2020, also is a divisional ofU.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, asU.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims thebenefit of priority to U.S. Provisional Application Nos. 61/631,313 and61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, eachentitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” andeach to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor.

This application also is a divisional of U.S. application Ser. No.16/824,572, now issued on Jun. 22, 2021, as U.S. Pat. No. 11,041,149,and filed Mar. 19, 2020, which is a continuation of U.S. applicationSer. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No.10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S.application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S.Pat. No. 9,447,401, filed on Dec. 28, 2012, which claims the benefit ofpriority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208,filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and eachentitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.”

U.S. application Ser. No. 16/824,572, now issued on Jun. 22, 2021, asU.S. Pat. No. 11,041,149, and filed Mar. 19, 2020, also is a divisionalof U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, asU.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims thebenefit of priority to U.S. Provisional Application Nos. 61/631,313 and61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, andeach entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USESTHEREOF,” and each to Ge Wei, H. Michael Shepard, Qiping Zhao and RobertJames Connor.

This application is a continuation of U.S. application Ser. No.15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400,entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” andfiled on Aug. 2, 2016, to Ge Wei, H. Michael Shepard, Qiping Zhao andRobert James Connor, which is a divisional of U.S. application Ser. No.13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, andfiled on Dec. 28, 2012, which claims the benefit of priority to U.S.Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec.30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.”

This application also is a divisional of U.S. application Ser. No.13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401,entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” andfiled on Dec. 28, 2012, to Ge Wei, H. Michael Shepard, Qiping Zhao andRobert James Connor, which claims the benefit of priority to U.S.Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec.30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.”

This application also is related to International PCT Application SerialNo. PCT/US2012/072182, filed Dec. 28, 2012, entitled “PH20 POLYPEPTIDEVARIANTS, FORMULATIONS AND USES THEREOF,” which also claims priority toU.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed onDec. 30, 2011, and Nov. 1, 2012, respectively.

The subject matter of each of the above-noted applications and patentsis incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Dec. 15, 2022, is 1,632 kilobytes insize, and is titled 3087Hseq001.xml.

FIELD OF THE INVENTION

Modified PH20 hyaluronidase polypeptides, including modifiedpolypeptides that exhibit increased stability and/or increased activity,are provided. Also provided are compositions and formulations and usesthereof.

BACKGROUND

Hyaluronan (hyaluronic acid; HA) is a polypeptide that is found in theextracellular matrix of many cells, especially in soft connectivetissues. HA also is found predominantly in skin, cartilage, and insynovial fluid in mammals. Hyaluronan also is the main constituent ofthe vitreous of the eye. HA has a role in various physiologicalprocesses, such as in water and plasma protein homeostasis (Laurent T Cet al. (1992) FASEB J 6: 2397-2404)). Certain diseases are associatedwith expression and/or production of hyaluronan. Hyaluronan-degradingenzymes, such as hyaluronidases, are enzymes that degrade hyaluronan. Bycatalyzing HA degradation, hyaluronan-degrading enzymes (e.g.,hyaluronidases) can be used to treat diseases or disorders associatedwith accumulation of HA or other glycosaminoglycans. Also, since HA is amajor component of the interstitial barrier, hyaluronan-degradingenzymes (e.g., hyaluronidase) increase tissue permeability and thereforecan be used to increase the dispersion and delivery of therapeuticagents. Various hyaluronidases have been used therapeutically (e.g.,hyaluronidase sold under the trademarks Hydase® (bovine testicularhyaluronidase), Vitrase® (ovine hyaluronidase), and Wydase® (bovinehyaluronidase)), typically as dispersing and spreading agents incombination with other therapeutic agents. Many of these are ovine orbovine forms, which can be immunogenic for treatment of humans. Improvedhyaluronan-degrading enzymes, such as hyaluronidases, and compositionsthereof that can be used for treatment are needed.

SUMMARY

Provided are modified PH20 polypeptides that have an altered property orproperties compared to the PH20 polypeptide that do not have themodification(s). The modifications include amino acid replacement,deletion and/or insertions. Detailed structure/function of virtuallyeach amino acid in a PH20 polypeptide is provided herein, as well as theidentification of residues and loci that contribute to alteration of aproperty, such as stability in particular conditions, is provided.Hence, provided are modified PH20 polypeptides that contain one or moreamino acid replacements that result in a PH20 polypeptide that retainsactivity and/or exhibits increased or altered stability under a varietyof conditions. Activity retained can be, for example, hyaluronidaseactivity that is as least about 40% or more of the PH20 polypeptide thatdoes not include the replacement. Exemplary modifications are amino acidreplacements. For purposes herein, amino acid replacements are denotedby the single amino acid letter followed by the corresponding amino acidposition in SEQ ID NO:3 in which the replacement occurs. Single aminoacid abbreviations for amino acid residues are well known to a skilledartisan (see e.g. Table 1), and are used herein throughout thedescription and examples. For example, replacement with P at a positioncorresponding to position 204 in a PH20 polypeptide with reference toamino acid residue positions set forth in SEQ ID NO:3 means that thereplacement encompasses F204P in a PH20 polypeptide set forth in SEQ IDNO:3, or the same replacement at the corresponding position in anotherPH20 polypeptide.

Provided are modified PH20 polypeptides that contain at least one aminoacid replacement in a PH20 polypeptide, whereby the modified PH20polypeptide exhibits increased stability compared to the PH20polypeptide not containing the amino acid replacement. Increasedstability can be manifested as increased resistance to one or moreprotein conditions that are denaturing to proteins. The stability ofmodified and unmodified PH20 is compared under the same conditions.Exemplary protein denaturation (or denaturing, used interchangeablyherein) conditions include, but are not limited to, elevated temperaturegreater than 30° C. or about 30° C., agitation, low salt, includingessentially or substantially or no salt, and presence of excipients thattend to denature proteins. Exemplary of such excipients areantiadherent(s), binder(s), coating(s), filler(s) and diluent(s),flavor(s), color(s), lubricant(s), glidant(s), preservative(s),detergent(s), sorbent(s) and combinations thereof.

The modified PH20 polypeptide can be one in which the unmodified formthereof has at least about 68% sequence identity to SEQ ID NO: 3 andfurther contains modifications that alter stability and/or can be a PH20polypeptide that includes as many as about up to 100, 110, 120, 130, 150amino acid differences from PH20 but retains enzymatic activity,particularly, at least about 40% of the activity of the unmodified PH20polypeptide and exhibits increased stability, such as stability underdenaturing conditions. Thus, included are modified PH20 polypeptidesthat have at least 68% or about 68% amino acid sequence identity to thesequence of amino acids set forth in SEQ ID NO:3. Included are modifiedPH20 polypeptides that have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acidsequence identity to the sequence of amino acids set forth in SEQ IDNO:3. Exemplary of such modified PH20 polypeptides are polypeptides thatcontain amino acid replacement(s) in a PH20 polypeptide that containsthe sequence of amino acid residues as set forth in any of SEQ ID NOs:3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, 870 or a sequence ofamino acids that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs:3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, or 870.

For example, provided herein is a modified PH20 polypeptide thatexhibits increased stability containing an amino acid replacement in aPH20 polypeptide that confers the increased stability, wherein increasedstability is manifested as increased resistance to denaturation in thepresence of one or more protein denaturation conditions, stability isincreased compared to the PH20 polypeptide not containing the amino acidreplacement, and the unmodified PH20 polypeptide consists of thesequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminaltruncated fragment thereof that is a soluble PH20 polypeptide or has atleast 85% sequence identity thereto. As above, the modified PH20polypeptide that exhibits increased stability exhibits increasedstability to a denaturation condition that is temperature greater thanor about 30° C.; agitation; low or no a salt; or presence of anexcipient or a denaturing agent, such as an antiadherent(s), binder(s),coating(s), filler(s) and diluent(s), flavor(s), color(s), lubricant(s),glidant(s), preservative(s), detergent(s), sorbent(s) or sweetener(s)and a combination thereof, and in particular a preservative. In someexamples of such modified PH20 polypeptides that exhibit increasedstability, the denaturation condition is temperature greater than 30°C., and the modified PH20 polypeptide exhibits greater hyaluronidaseactivity at the temperature compared to the unmodified PH20 polypeptidenot containing the amino acid replacement(s) where the activities arecompared under the same conditions. In other examples, the proteindenaturation condition is the presence of low concentrations of salt ofless than 100 mM, and the modified PH20 polypeptide exhibits increasedhyaluronidase activity in the presence of low concentrations of saltcompared to the unmodified PH20 polypeptide not containing the aminoacid replacement(s) where the activities are compared under the sameconditions.

In any of the above examples of a modified PH20 polypeptide thatexhibits increased stability, stability can be assessed based on avariety of parameters including hyaluronidase activity, solubility,aggregation and/or crystallization. Stability can be assessed in thepresence of a denaturing condition. When stability of two or morepolypeptides is compared, stability is assessed under the sameconditions. In some instances, among the PH20 polypeptides providedherein, the modified PH20 polypeptide exhibits at least 120%, 130%,135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%,500%, 1500%, 2000%, 3000%, 4000%, 5000% or more of the hyaluronidaseactivity of the PH20 polypeptide not containing the amino acidreplacement(s).

In any of the above examples of a modified PH20 polypeptide thatexhibits increased stability, denaturing conditions include the presenceof excipients that denature proteins. Exemplary of such conditions isthe presence of a preservative, such as a phenolic preservative.Provided are modified PH20 polypeptides that exhibit increased stabilityin the presence of an anti-microbial effective amount of one or morephenolic preservatives. An anti-microbial effective amount is the totalamount of one or more phenolic preservative agents, which can beexpressed as a percentage (%) of mass concentration (w/v) that is or isbetween (or at least about or at about) 0.05% to 0.6%, 0.1% to 0.4%,0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to0.3% or 0.3% to 0.4%, inclusive. Exemplary phenolic preservativesinclude, but are not limited to, phenol, metacresol (m-cresol), benzylalcohol, and a paraben, such as methylparaben propylparaben, m-cresol,phenol or m-cresol and phenol. Exemplary of the stability achieved byprovided modified PH20 polypeptides are those that exhibit at least 15%or about 15% of the hyaluronidase activity for at least 4 hours in thepresence of preservative(s) compared to the modified PH20 polypeptide inabsence of preservative. Activity is compared under the same conditionsexcept for the presence of preservative(s). For example, provided aremodified PH20 polypeptides that exhibit at least (or at least about)16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the hyaluronidase activityin the presence of a phenolic preservative(s) compared to absence of thesame preservative(s). Thus, provided, among the modified PH20polypeptides provided herein, are PH20 polypeptides that, by virtue ofamino acid replacement(s), are phenophilic compared to PH20 polypeptideswithout such replacement. Included are modified PH20 polypeptides wherethe hyaluronidase activity is exhibited after at least 5 hours, 6 hours,7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks or more in thepresence of the preservative(s) compared to the hyaluronidase activityof the modified PH20 polypeptide in the absence of preservative for thesame time period and under the same conditions except for the presenceof preservative(s).

In examples of a modified PH20 polypeptide that exhibits increasedstability to a phenolic preservative, increased stability in a phenolicpreservative can be exhibited under temperature conditions that includeany temperature between, for example, 0° C. and 40° C., such as betweenor about between 0° C. to 40° C., 2° C. to 6° C., 24° C. to 32° C. and35° C. to 40° C. Exemplary polypeptides exhibit increased stability attemperatures of between or about between 30° C. to 45° C., 35° C. to 45°C., 30° C. to 37° C., 35° C. to 37° C. or 37° C. to 42° C., eachinclusive. The particular modified PH20 polypeptide and conditionsdepend upon the intended formulation, conditions to which theformulation will be exposed and/or intended application.

Particular and exemplary modified PH20 polypeptides that exhibitincreased stability, such as increased stability to a phenolicpreservative, include those that contain a single amino acidmodification, such as a replacement, and combinations of modifications,such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and more modifications.These include modified PH20 polypeptides that contain one or more aminoacid replacements, where at least one replacement is at an amino acidposition corresponding (i.e., by alignment) to a position selected fromamong 10, 12, 20, 22, 26, 34, 36, 46, 50, 52, 58, 68, 70, 74, 82, 83,84, 86, 97, 127, 131, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196,204, 205, 206, 213, 219, 234, 237, 238, 240, 249, 261, 267, 277, 279,291, 309, 310, 314, 315, 317, 318, 347, 367, 375, 376, 399, 401, 407,416, 419, 421, 431, 433, 439, 440, 443 or 445 with reference to aminoacid positions set forth in SEQ ID NO:3, wherein corresponding aminoacid positions are identified by alignment of the PH20 polypeptide withthe polypeptide set forth in SEQ ID NO:3. Exemplary of suchmodifications are at least one amino acid replacement selected fromamong replacement with: glycine (G) at a position corresponding toposition 10; K at a position corresponding to position 12; S at aposition corresponding to position 20; T at a position corresponding toposition 22; M at a position corresponding to position 26; W at aposition corresponding to position 34; N at a position corresponding toposition 36; L at a position corresponding to position 46; M at aposition corresponding to position 50; T at a position corresponding toposition 52; S at a position corresponding to position 52; C at aposition corresponding to position 58; K at a position corresponding toposition 58; Rat a position corresponding to position 58; N at aposition corresponding to position 58; Y at a position corresponding toposition 58; P at a position corresponding to position 58; H at aposition corresponding to position 58; P at a position corresponding toposition 68; V at a position corresponding to position 70; E at aposition corresponding to position 74; L at a position corresponding toposition 82; N at a position corresponding to position 82; V at aposition corresponding to position 83; Q at a position corresponding toposition 83; S at a position corresponding to position 83; G at aposition corresponding to position 83; N at a position corresponding toposition 84; A at a position corresponding to position 86; K at aposition corresponding to position 86; E at a position corresponding toposition 97; L at a position corresponding to position 97; R at aposition corresponding to position 127; Rat a position corresponding toposition 131; L at a position corresponding to position 138; K at aposition corresponding to position 142; N at a position corresponding toposition 142; P at a position corresponding to position 142; S at aposition corresponding to position 142; T at a position corresponding toposition 142; G at a position corresponding to position 143; K at aposition corresponding to position 143; T at a position corresponding toposition 144; Q at a position corresponding to position 166; T at aposition corresponding to position 166; L at a position corresponding toposition 169; G at a position corresponding to position 174; N at aposition corresponding to position 174; Q at a position corresponding toposition 193; T at a position corresponding to position 195; N at aposition corresponding to position 195; E at a position corresponding toposition 196; Rat a position corresponding to position 196; P at aposition corresponding to position 204; A at a position corresponding toposition 205; E at a position corresponding to position 205; I at aposition corresponding to position 206; A at a position corresponding toposition 213; I at a position corresponding to position 219; M at aposition corresponding to position 234; T at a position corresponding toposition 237; H at a position corresponding to position 238; Q at aposition corresponding to position 240; V at a position corresponding toposition 249; A at a position corresponding to position 261; K at aposition corresponding to position 261; Tat a position corresponding toposition 267; K at a position corresponding to position 277; H at aposition corresponding to position 279; V at a position corresponding toposition 279; V at a position corresponding to position 291; E at aposition corresponding to position 309; Q at a position corresponding toposition 310; Y at a position corresponding to position 314; Y at aposition corresponding to position 315; N at a position corresponding toposition 317; W at a position corresponding to position 317; D at aposition corresponding to position 318; G at a position corresponding toposition 347; A at a position corresponding to position 367; R at aposition corresponding to position 375; Rat a position corresponding toposition 376; V at a position corresponding to position 399; E at aposition corresponding to position 401; A at a position corresponding toposition 407; L at a position corresponding to position 416; K at aposition corresponding to position 419; H at a position corresponding toposition 421; E at a position corresponding to position 431; Tat aposition corresponding to position 433; V at a position corresponding toposition 433; C at a position corresponding to position 439; P at aposition corresponding to position 440; G at a position corresponding toposition 443; N at a position corresponding to position 445, withreference to amino acid residue positions set forth in SEQ ID NO:3. Forexample, the modified PH20 polypeptide can contain at least one aminoacid replacement selected from among replacement with: T at a positioncorresponding to position 52, K at a position corresponding to position58, Rat a position corresponding to position 58, P at a positioncorresponding to position 68, V at a position corresponding to position83, P at a position corresponding to position 204, A at a positioncorresponding to position 261, Tat a position corresponding to position267, K at a position corresponding to position 277 and H at a positioncorresponding to position 421, with reference to amino acid residuepositions set forth in SEQ ID NO:3. An exemplary modified PH20polypeptide is one that includes P (or a conservative amino acidthereto) at a position corresponding to position 204 in a PH20polypeptide with reference to amino acid residue positions set forth inSEQ ID NO:3.

Thus, provided herein are modified PH20 polypeptides that exhibitincreased stability in the presence of a phenolic preservativecontaining an amino acid replacement in a PH20 polypeptide that confersthe increased stability, wherein stability is increased compared to theunmodified polypeptide without the amino acid replacement, and theunmodified PH20 polypeptide has the sequence of amino acids set forth inSEQ ID NO: 7 or is a C-terminal truncated fragment thereof that is asoluble PH20 polypeptide or has at least 85% sequence identity thereto.For example, the unmodified PH20 polypeptide is a soluble PH20polypeptide that has the sequence of amino acids set forth in any of SEQID NOs: 3 or 32-66. In particular examples, the modified PH20polypeptide has at least 85% sequence identity to SEQ ID NO:3. In any ofsuch examples of a modified PH20 polypeptide, the polypeptide contains1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75 or more amino acid replacements. In examples herein, the modifiedPH20 polypeptide is a human PH20. The modified PH20 polypeptide exhibitsstability in the presence of phenolic preservatives if it exhibits atleast 15% of the hyaluronidase activity in the presence of apreservative(s) for at least 4 hours compared to the hyaluronidaseactivity in the absence of the phenolic preservative(s), wherein theactivity is compared under the same conditions except for the presenceof the phenolic preservative(s). In any of the above examples, themodified PH20 polypeptide is stable in the presence of an of ananti-microbial effective amount of one or more phenolic preservatives,such as a total amount of one or more phenolic preservative agents as apercentage (%) of mass concentration (w/v) that is from or from about0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4%, inclusive. Thephenolic preservative can be a phenol, metacresol (m-cresol), benzylalcohol or a paraben, such as m-cresol, phenol, or m-cresol and phenol.The amino acid replacement can be at amino acid residue 204, 58, 10, 12,20, 22, 26, 34, 36, 46, 50, 52, 68, 70, 74, 82, 83, 84, 86, 97, 127,131, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196, 205, 206, 213,219, 234, 237, 238, 240, 249, 261, 267, 277, 279, 291, 309, 310, 314,315, 317, 318, 347, 367, 375, 376, 399, 401, 407, 416, 419, 421, 431,433, 439, 440, 443 or 445 with reference to amino acid positions setforth in SEQ ID NO:3, wherein corresponding amino acid positions areidentified by alignment of the PH20 polypeptide with the polypeptide setforth in SEQ ID NO:3. For example, the amino acid replacement is G at aposition corresponding to position 10; K at a position corresponding toposition 12; S at a position corresponding to position 20; T at aposition corresponding to position 22; M at a position corresponding toposition 26; W at a position corresponding to position 34; N at aposition corresponding to position 36; L at a position corresponding toposition 46; M at a position corresponding to position 50; T at aposition corresponding to position 52; S at a position corresponding toposition 52; C at a position corresponding to position 58; K at aposition corresponding to position 58; Rat a position corresponding toposition 58; N at a position corresponding to position 58; Y at aposition corresponding to position 58; P at a position corresponding toposition 58; H at a position corresponding to position 58; P at aposition corresponding to position 68; V at a position corresponding toposition 70; E at a position corresponding to position 74; L at aposition corresponding to position 82; N at a position corresponding toposition 82; V at a position corresponding to position 83; Q at aposition corresponding to position 83; S at a position corresponding toposition 83; G at a position corresponding to position 83; N at aposition corresponding to position 84; A at a position corresponding toposition 86; K at a position corresponding to position 86; E at aposition corresponding to position 97; L at a position corresponding toposition 97; R at a position corresponding to position 127; Rat aposition corresponding to position 131; L at a position corresponding toposition 138; K at a position corresponding to position 142; N at aposition corresponding to position 142; P at a position corresponding toposition 142; S at a position corresponding to position 142; T at aposition corresponding to position 142; G at a position corresponding toposition 143; K at a position corresponding to position 143; T at aposition corresponding to position 144; Q at a position corresponding toposition 166; T at a position corresponding to position 166; L at aposition corresponding to position 169; G at a position corresponding toposition 174; N at a position corresponding to position 174; Q at aposition corresponding to position 193; T at a position corresponding toposition 195; N at a position corresponding to position 195; E at aposition corresponding to position 196; Rat a position corresponding toposition 196; P at a position corresponding to position 204; A at aposition corresponding to position 205; E at a position corresponding toposition 205; I at a position corresponding to position 206; A at aposition corresponding to position 213; I at a position corresponding toposition 219; M at a position corresponding to position 234; T at aposition corresponding to position 237; H at a position corresponding toposition 238; Q at a position corresponding to position 240; V at aposition corresponding to position 249; A at a position corresponding toposition 261; K at a position corresponding to position 261; T at aposition corresponding to position 267; K at a position corresponding toposition 277; H at a position corresponding to position 279; V at aposition corresponding to position 279; V at a position corresponding toposition 291; E at a position corresponding to position 309; Q at aposition corresponding to position 310; Y at a position corresponding toposition 314; Y at a position corresponding to position 315; N at aposition corresponding to position 317; W at a position corresponding toposition 317; D at a position corresponding to position 318; G at aposition corresponding to position 347; A at a position corresponding toposition 367; R at a position corresponding to position 375; Rat aposition corresponding to position 376; V at a position corresponding toposition 399; E at a position corresponding to position 401; A at aposition corresponding to position 407; L at a position corresponding toposition 416; K at a position corresponding to position 419; H at aposition corresponding to position 421; E at a position corresponding toposition 431; T at a position corresponding to position 433; V at aposition corresponding to position 433; C at a position corresponding toposition 439; P at a position corresponding to position 440; G at aposition corresponding to position 443; or N at a position correspondingto position 445, with reference to amino acid residue positions setforth in SEQ ID NO:3. In particular, the amino acid replacement is T ata position corresponding to position 52, K at a position correspondingto position 58, Rat a position corresponding to position 58, P at aposition corresponding to position 68, V at a position corresponding toposition 83, P at a position corresponding to position 204, A at aposition corresponding to position 261, T at a position corresponding toposition 267, K at a position corresponding to position 277 or H at aposition corresponding to position 421, with reference to amino acidresidue positions set forth in SEQ ID NO:3, such as replacement with Pat a position corresponding to position 204 or Rat a positioncorresponding to position 58. The modified PH20 polypeptide thatexhibits increased stability to phenolic preservatives can besubstantially purified or isolated. The modified PH20 polypeptide thatexhibits increased stability to phenolic preservatives can be modifiedby glycosylation, sialation, albumination, farnysylation, carboxylation,hydroxylation and phosphorylation, and generally is glycosylated,whereby the polypeptide contains at least an N-acetylglucosamine moietylinked to each of at least three asparagine (N) residues, such as atamino acid residues corresponding to amino acid residues 200, 333 and358 of SEQ ID NO:3. The modified PH20 polypeptide that exhibitsincreased stability to phenolic preservatives can be conjugated to apolymer, such as PEG or dextran and/or can be conjugated to a moietythat is a multimerization domain, a toxin, a detectable label or a drug.

Among modified PH20 polypeptides provided herein that exhibit increasedstability are those that exhibit increased hyaluronidase activity at theelevated temperature compared to the PH20 polypeptide not containing theamino acid replacement(s), such as at least 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or morehyaluronidase activity for at least 4 hours compared to the PH20polypeptide not containing the amino acid replacement(s). Also among thepolypeptides are those that exhibit activity, but also typically exhibitincreased stability or other property at elevated temperatures, such asa modified PH20 polypeptide that exhibits at least 95%, 96%, 97%, 98%,99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,300%, 400%, 500% of the hyaluronidase activity for at least 4 hours at atemperature of between or about between 32° C. to 37° C. compared to thehyaluronidase activity of the modified PH20 polypeptide at a temperatureof between or about between 2° C. to 8° C., where activity is comparedunder the same conditions except for the differences in temperature. Thehyaluronidase activity can be exhibited after at least 5 hours, 6 hours,7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks or more at elevatedtemperatures of between or about between 32° C. to 37° C. compared tothe hyaluronidase activity of the modified PH20 polypeptide at atemperature between or about between 2° C. to 8° C., where activity iscompared for the same time period and under the same conditions exceptfor the difference in temperature. Exemplary of such modifiedpolypeptides are those that contain at least one amino acid replacementat an amino acid position corresponding to a position selected fromamong 1, 11, 12, 14, 20, 26, 29, 34, 50, 58, 70, 82, 83, 84, 86, 87,140, 142, 143, 147, 152, 166, 167, 172, 174, 178, 193, 195, 206, 212,213, 219, 233, 237, 240, 267, 277, 291, 292, 309, 313, 314, 317, 318,347, 367, 368, 371, 374, 389, 392, 395, 396, 406, 419, 421, 439 and 443with reference to amino acid positions set forth in SEQ ID NO:3, whereincorresponding amino acid positions are identified by alignment of thePH20 polypeptide with the polypeptide set forth in SEQ ID NO:3.Exemplary mutations include, for example, replacement with Rat aposition corresponding to position 1; S at a position corresponding toposition 11; I at a position corresponding to position 12; V at aposition corresponding to position 14; S at a position corresponding toposition 20; M at a position corresponding to position 26; with R at aposition corresponding to position 29; W at a position corresponding toposition 34; M at a position corresponding to position 50; K at aposition corresponding to position 58; Q at a position corresponding toposition 58; Q at a position corresponding to position 58; V at aposition corresponding to position 70; L at a position corresponding toposition 82; Q at a position corresponding to position 83; Rat aposition corresponding to position 84; A at a position corresponding toposition 86; S at a position corresponding to position 87; K at aposition corresponding to position 140; S at a position corresponding toposition 142; T at a position corresponding to position 142; K at aposition corresponding to position 143; S at a position corresponding toposition 147; T at a position corresponding to position 152; T at aposition corresponding to position 166; D at a position corresponding toposition 167; A at a position corresponding to position 172; G at aposition corresponding to position 174; N at a position corresponding toposition 174; Rat a position corresponding to position 178; Q at aposition corresponding to position 193; T at a position corresponding toposition 195; I at a position corresponding to position 206; S at aposition corresponding to position 212; A at a position corresponding toposition 213; I at a position corresponding to position 219; G at aposition corresponding to position 233; Tat a position corresponding toposition 237; A at a position corresponding to position 240; Q at aposition corresponding to position 240; T at a position corresponding toposition 267; E at a position corresponding to position 277; S at aposition corresponding to position 291; H at a position corresponding toposition 292; V at a position corresponding to position 292; S at aposition corresponding to position 309; H at a position corresponding toposition 313; S at a position corresponding to position 314; I at aposition corresponding to position 317; T at a position corresponding toposition 317; W at a position corresponding to position 317; Rat aposition corresponding to position 318; G at a position corresponding toposition 347; A at a position corresponding to position 367; Rat aposition corresponding to position 368; S at a position corresponding toposition 371; P at a position corresponding to position 374; A at aposition corresponding to position 389; V at a position corresponding toposition 392; A at a position corresponding to position 395; H at aposition corresponding to position 396; N at a position corresponding toposition 406; H at a position corresponding to position 419; K at aposition corresponding to position 419; R at a position corresponding toposition 421; S at a position corresponding to position 421; A at aposition corresponding to position 439; C at a position corresponding toposition 439; and G at a position corresponding to position 443, withreference to amino acid positions set forth in SEQ ID NO:3. Inparticular examples provided herein, any of such modified PH20polypeptides contain a single amino acid modification, such as areplacement, and combinations of modifications, such as at least or 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 60, 70, 80, 90, 100 and more modifications. The modification, suchas replacement, can be in an unmodified PH20 polypeptide that has thesequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminaltruncated fragment thereof that is a soluble PH20 polypeptide, such asis set forth in any of SEQ ID NOs: 3 or 32-66, or has at least 85%sequence identity thereto. For example, any of such modified PH20polypeptides has at least 85% sequence identity to SEQ ID NO:3.

Also provided are modified PH20 polypeptides that exhibit increasedstability in low salt conditions, such as, for example, concentrationsof NaCl of less than 100 mM, such as, but not limited to concentrationsof NaCl less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25mM, 20 mM, 15 mM, 10 mM, 5 mM or less. Among the modified PH20polypeptides are those that exhibit increased hyaluronidase activity atlower concentrations of salt compared to the PH20 polypeptide notcontaining the amino acid replacement(s). Such activity includes, forexample, at least more than 100%, or at least 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or morehyaluronidase activity compared to the PH20 polypeptide not containingthe amino acid replacement(s). Exemplary of such modified PH20polypeptides are those that exhibit at least 60% of the hyaluronidaseactivity in low concentrations of salt, such as between or about between10 mM NaCl and 100 mM NaCl, inclusive (or comparable concentrations ofother salts or mixtures of salts), compared to the hyaluronidaseactivity of the modified PH20 polypeptide in 150 mM NaCl, whereactivities are compared under the same conditions except for thedifference in salt concentration. In particular examples providedherein, any of such modified PH20 polypeptides contain a single aminoacid modification, such as a replacement, and combinations ofmodifications, such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and moremodifications. The modification, such as replacement, can be in an theunmodified PH20 polypeptide that has the sequence of amino acids setforth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof thatis a soluble PH20 polypeptide, such as is set forth in any of SEQ IDNOs: 3 or 32-66, or has at least 85% sequence identity thereto. Forexample, any of such modified PH20 polypeptides has at least 85%sequence identity to SEQ ID NO:3.

Also provided are modified PH20 polypeptides that contain at least oneamino acid replacement in a PH20 polypeptide, where the modified PH20polypeptide exhibits increased hyaluronidase activity compared to thePH20 polypeptide not containing the amino acid replacement. Whencomparing activity among polypeptides, activity is compared under thesame conditions. Among these are polypeptides, where the unmodified PH20exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity tothe sequence of amino acids set forth in SEQ ID NO:3, or the resultingmodified PH20 exhibits such sequence identity to the sequence of aminoacids set forth in SEQ ID NO:3. Exemplary of such modified PH20polypeptides are any that contain an amino acid replacement(s) in thesequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 10, 12,14, 24, 32-66, 69, or 72, or a sequence of amino acids that is at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identical to any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66,69, or 72. The amino acid replacement(s) also can be made in thesequence of amino acids set forth in any of SEQ ID NOs: 857, 859, 861 or870, or a sequence of amino acids that is at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical toany of SEQ ID NOs: 857, 859, 861 or 870. In particular, provided aremodified PH20 polypeptides that contain an amino acid replacement in thesequence of amino acids set forth in SEQ ID NOs: 3, 7, 32-66, 69 or 72.Among the modified PH20 polypeptides are those that that exhibit atleast 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%,300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% or more of thehyaluronidase activity of the PH20 polypeptide not containing the aminoacid replacement. Activity can be assessed at any temperature, inparticular such activity is present when the hyaluronidase is exposed toa temperature that is at a temperature between or about between 2° C. to8° C. These modified PH20 polypeptides contain at least one amino acidreplacement at an amino acid position corresponding to a positionselected from among 1, 12, 15, 24, 26, 27, 29, 30, 31, 32, 33, 37, 39,46, 48, 52, 58, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 84, 86, 87, 92,93, 94, 97, 118, 120, 127, 131, 135, 141, 142, 147, 148, 150, 151, 152,155, 156, 163, 164, 165, 166, 169, 170, 174, 198, 206, 209, 212, 213,215, 219, 233, 234, 236, 238, 247, 257, 259, 260, 261, 263, 269, 271,272, 276, 277, 278, 282, 291, 293, 305, 308, 309, 310, 313, 315, 317,318, 324, 325, 326, 328, 347, 353, 359, 371, 377, 380, 389, 392, 395,399, 405, 407, 409, 410, 418, 419, 421, 425, 431, 433, 436, 437, 438,439, 440, 441, 442, 443, 445, 446 and 447 with reference to amino acidpositions set forth in SEQ ID NO:3, wherein corresponding amino acidpositions are identified by alignment of the PH20 polypeptide with thepolypeptide set forth in SEQ ID NO:3. Exemplary modifications include atleast one amino acid replacement selected from among replacement with:histidine (H) at a position corresponding to position 1; Q at a positioncorresponding to position 1; E at a position corresponding to position12; T at a position corresponding to position 12; V at a positioncorresponding to position 15; E at a position corresponding to position24; H at a position corresponding to position 24; E at a positioncorresponding to position 26; K at a position corresponding to position26; K at a position corresponding to position 27; R at a positioncorresponding to position 27; E at a position corresponding to position29; I at a position corresponding to position 29; L at a positioncorresponding to position 29; M at a position corresponding to position29; P at a position corresponding to position 29; S at a positioncorresponding to position 29; V at a position corresponding to position29; G at a position corresponding to position 30; H at a positioncorresponding to position 30; K at a position corresponding to position30; M at a position corresponding to position 30; R at a positioncorresponding to position 30; S at a position corresponding to position30; A at a position corresponding to position 31; C at a positioncorresponding to position 31; H at a position corresponding to position31; I at a position corresponding to position 31; K at a positioncorresponding to position 31; L at a position corresponding to position31; P at a position corresponding to position 31; R at a positioncorresponding to position 31; S at a position corresponding to position31; T at a position corresponding to position 31; V at a positioncorresponding to position 31; F at a position corresponding to position32; G at a position corresponding to position 32; H at a positioncorresponding to position 32; W at a position corresponding to position33; F at a position corresponding to position 37; N at a positioncorresponding to position 39; T at a position corresponding to position39; R at a position corresponding to position 46; F at a positioncorresponding to position 48; H at a position corresponding to position48; N at a position corresponding to position 48; Q at a positioncorresponding to position 52; K at a position corresponding to position58; Q at a position corresponding to position 58; W at a positioncorresponding to position 63; V at a position corresponding to position67; H at a position corresponding to position 68; Q at a positioncorresponding to position 68; A at a position corresponding to position69; C at a position corresponding to position 69; F at a positioncorresponding to position 69; G at a position corresponding to position69; I at a position corresponding to position 69; L at a positioncorresponding to position 69; M at a position corresponding to position69; P at a position corresponding to position 69; R at a positioncorresponding to position 69; W at a position corresponding to position69; Y at a position corresponding to position 69; A at a positioncorresponding to position 70; C at a position corresponding to position70; F at a position corresponding to position 70; G at a positioncorresponding to position 70; H at a position corresponding to position70; K at a position corresponding to position 70; L at a positioncorresponding to position 70; N at a position corresponding to position70; P at a position corresponding to position 70; R at a positioncorresponding to position 70; S at a position corresponding to position70; T at a position corresponding to position 70; V at a positioncorresponding to position 70; Rat a position corresponding to position71; S at a position corresponding to position 71; M at a positioncorresponding to position 72; Q at a position corresponding to position72; H at a position corresponding to position 73; L at a positioncorresponding to position 73; W at a position corresponding to position73; A at a position corresponding to position 74; C at a positioncorresponding to position 74; G at a position corresponding to position74; N at a position corresponding to position 74; P at a positioncorresponding to position 74; R at a position corresponding to position74; S at a position corresponding to position 74; V at a positioncorresponding to position 74; W at a position corresponding to position74; F at a position corresponding to position 75; L at a positioncorresponding to position 75; Rat a position corresponding to position75; T at a position corresponding to position 75; G at a positioncorresponding to position 84; Rat a position corresponding to position84; A at a position corresponding to position 86; C at a positioncorresponding to position 87; T at a position corresponding to position87; Y at a position corresponding to position 87; C at a positioncorresponding to position 92; I at a position corresponding to position93; L at a position corresponding to position 93; Rat a positioncorresponding to position 93; T at a position corresponding to position93; Rat a position corresponding to position 94; G at a positioncorresponding to position 97; Q at a position corresponding to position118; F at a position corresponding to position 120; V at a positioncorresponding to position 120; Y at a position corresponding to position120; H at a position corresponding to position 127; N at a positioncorresponding to position 127; G at a position corresponding to position131; Rat a position corresponding to position 131; V at a positioncorresponding to position 131; D at a position corresponding to position135; G at a position corresponding to position 135; Rat a positioncorresponding to position 135, with H at a position corresponding toposition 141; Y at a position corresponding to position 141; Rat aposition corresponding to position 142; R at a position corresponding toposition 147; V at a position corresponding to position 147; K at aposition corresponding to position 148; G at a position corresponding toposition 150; K at a position corresponding to position 151; L at aposition corresponding to position 151; M at a position corresponding toposition 151; Q at a position corresponding to position 151; R at aposition corresponding to position 151; R at a position corresponding toposition 152; G at a position corresponding to position 155; K at aposition corresponding to position 155; D at a position corresponding toposition 156; A at a position corresponding to position 163; E at aposition corresponding to position 163; K at a position corresponding toposition 163; Rat a position corresponding to position 163; M at aposition corresponding to position 164; D at a position corresponding toposition 165; N at a position corresponding to position 165; A at aposition corresponding to position 166; F at a position corresponding toposition 166; H at a position corresponding to position 166; L at aposition corresponding to position 166; Q at a position corresponding toposition 166; Rat a position corresponding to position 166; T at aposition corresponding to position 166; Y at a position corresponding toposition 166; L at a position corresponding to position 169; Rat aposition corresponding to position 170; K at a position corresponding toposition 174; D at a position corresponding to position 198; K at aposition corresponding to position 206; L at a position corresponding toposition 206; N at a position corresponding to position 212; M at aposition corresponding to position 213; N at a position corresponding toposition 213; M at a position corresponding to position 215; S at aposition corresponding to position 219; K at a position corresponding toposition 233; Rat a position corresponding to position 233; M at aposition corresponding to position 234; R at a position corresponding toposition 236; E at a position corresponding to position 237; S at aposition corresponding to position 238; I at a position corresponding toposition 247; T at a position corresponding to position 257; P at aposition corresponding to position 259; Y at a position corresponding toposition 260; K at a position corresponding to position 261; N at aposition corresponding to position 261; K at a position corresponding toposition 263; Rat a position corresponding to position 263; A at aposition corresponding to position 269; L at a position corresponding toposition 271; M at a position corresponding to position 271; T at aposition corresponding to position 272; D at a position corresponding toposition 276; S at a position corresponding to position 276; Y at aposition corresponding to position 276; K at a position corresponding toposition 277; R at a position corresponding to position 277; T at aposition corresponding to position 277; H at a position corresponding toposition 278; K at a position corresponding to position 278; N at aposition corresponding to position 278; R at a position corresponding toposition 278; S at a position corresponding to position 278; T at aposition corresponding to position 278; Y at a position corresponding toposition 278; M at a position corresponding to position 282; V at aposition corresponding to position 291; A at a position corresponding toposition 293; C at a position corresponding to position 293; F at aposition corresponding to position 293; M at a position corresponding toposition 293; P at a position corresponding to position 293; Q at aposition corresponding to position 293; V at a position corresponding toposition 293; E at a position corresponding to position 305; G at aposition corresponding to position 308; N at a position corresponding toposition 308; E at a position corresponding to position 309; L at aposition corresponding to position 309; N at a position corresponding toposition 309; Q at a position corresponding to position 309; R at aposition corresponding to position 309; T at a position corresponding toposition 309; A at a position corresponding to position 310; G at aposition corresponding to position 310; K at a position corresponding toposition 313; Rat a position corresponding to position 313; H at aposition corresponding to position 315; I at a position corresponding toposition 317; K at a position corresponding to position 317; Rat aposition corresponding to position 317; M at a position corresponding toposition 318; H at a position corresponding to position 320; K at aposition corresponding to position 320; Rat a position corresponding toposition 320; R at a position corresponding to position 324; A at aposition corresponding to position 325; D at a position corresponding toposition 325; E at a position corresponding to position 325; G at aposition corresponding to position 325; H at a position corresponding toposition 325; K at a position corresponding to position 325; M at aposition corresponding to position 325; N at a position corresponding toposition 325; Q at a position corresponding to position 325; S at aposition corresponding to position 325; V at a position corresponding toposition 326; I at a position corresponding to position 328; K at aposition corresponding to position 328; L at a position corresponding toposition 328; S at a position corresponding to position 328; Y at aposition corresponding to position 328; G at a position corresponding toposition 347; S at a position corresponding to position 347; V at aposition corresponding to position 353; with T at a positioncorresponding to position 359; R at a position corresponding to position371; P at a position corresponding to position 377; Tat a positioncorresponding to position 377; W at a position corresponding to position380; Y at a position corresponding to position 380; K at a positioncorresponding to position 389; M at a position corresponding to position392; Rat a position corresponding to position 395; M at a positioncorresponding to position 399; T at a position corresponding to position399; W at a position corresponding to position 399; G at a positioncorresponding to position 405; D at a position corresponding to position407; Q at a position corresponding to position 407; A at a positioncorresponding to position 409; Q at a position corresponding to position409; T at a position corresponding to position 410; P at a positioncorresponding to position 418; F at a position corresponding to position419; I at a position corresponding to position 419; K at a positioncorresponding to position 419; Rat a position corresponding to position419; S at a position corresponding to position 419; H at a positioncorresponding to position 421; K at a position corresponding to position421; N at a position corresponding to position 421; Q at a positioncorresponding to position 421; R at a position corresponding to position421; S at a position corresponding to position 421; K at a positioncorresponding to position 425; A at a position corresponding to position431; H at a position corresponding to position 431; K at a positioncorresponding to position 431; Q at a position corresponding to position431; R at a position corresponding to position 431; S at a positioncorresponding to position 431; V at a position corresponding to position431; L at a position corresponding to position 433; Rat a positioncorresponding to position 433; T at a position corresponding to position433; V at a position corresponding to position 433; K at a positioncorresponding to position 436; I at a position corresponding to position437; M at a position corresponding to position 437; T at a positioncorresponding to position 438; V at a position corresponding to position439; H at a position corresponding to position 440; R at a positioncorresponding to position 440; F at a position corresponding to position441; Rat a position corresponding to position 442; A at a positioncorresponding to position 443; M at a position corresponding to position443; M at a position corresponding to position 445; P at a positioncorresponding to position 445; A at a position corresponding to position446; D at a position corresponding to position 447; N at a positioncorresponding to position 447; and/or with Q at a position correspondingto position 447, with reference to amino acid positions set forth in SEQID NO:3.

Among the polypeptides that exhibit increased hyaluronidase activity arethose that exhibit at least 2.0-fold of the hyaluronidase activity ofthe PH20 polypeptide not containing the amino acid replacement. Forexample, among these are modified PH20 polypeptides that contain atleast one amino acid replacement at an amino acid position correspondingto a position selected from among 24, 29, 31, 48, 58, 69, 70, 75, 84,97, 165, 166, 271, 278, 317, 320, 325 and 326 with reference topositions set forth in SEQ ID NO:3, wherein corresponding amino acidpositions are identified by alignment of the PH20 polypeptide with thepolypeptide set forth in SEQ ID NO:3, such as modified PH20 polypeptidesthat contain at least one amino acid replacement selected from amongreplacement with: E at a position corresponding to position 24; E at aposition corresponding to position 29; V at a position corresponding toposition 31; N at a position corresponding to position 48; K at aposition corresponding to position 58; Q at a position corresponding toposition 58; A at a position corresponding to position 69; F at aposition corresponding to position 69; G at a position corresponding toposition 69; P at a position corresponding to position 69; R at aposition corresponding to position 69; A at a position corresponding toposition 70; F at a position corresponding to position 70; G at aposition corresponding to position 70; H at a position corresponding toposition 70; H at a position corresponding to position 70; N at aposition corresponding to position 70; R at a position corresponding toposition 70; T at a position corresponding to position 70; V at aposition corresponding to position 70; L at a position corresponding toposition 75; T at a position corresponding to position 75; G at aposition corresponding to position 84; G at a position corresponding toposition 97; D at a position corresponding to position 165; L at aposition corresponding to position 166; Rat a position corresponding toposition 166; T at a position corresponding to position 166; L at aposition corresponding to position 271; H at a position corresponding toposition 278; Rat a position corresponding to position 278; K at aposition corresponding to position 317; K at a position corresponding toposition 320; E at a position corresponding to position 325, with G at aposition corresponding to position 325; K at a position corresponding toposition 325; N at a position corresponding to position 325; Q at aposition corresponding to position 325; and V at a positioncorresponding to position 326; with reference to amino acid positionsset forth in SEQ ID NO:3.

Among any of the polypeptides provided herein that exhibit increasedhyaluronidase activity, any of such modified PH20 polypeptides contain asingle amino acid modification, such as a replacement, and combinationsof modifications, such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 andmore modifications. The modification, such as replacement, can be in anunmodified PH20 polypeptide that has the sequence of amino acids setforth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof thatis a soluble PH20 polypeptide, such as is set forth in any of SEQ IDNOs: 3 or 32-66, or has at least 85% sequence identity thereto. Forexample, any of such modified PH20 polypeptides has at least 85%sequence identity to SEQ ID NO:3.

Also provided are modified PH20 polypeptides that contain at least oneamino acid replacement in the PH20 polypeptide whose sequence is setforth in SEQ ID NO:7, a C-terminally truncated fragment thereof, asoluble fragment thereof, or in a PH20 polypeptide that has a sequenceof amino acids that is at least 91% identical to the sequence of aminoacids set forth in SEQ ID NO:7, where at least one amino replacement(s)is at an amino acid position corresponding to a position selected fromamong 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 24,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 58, 59, 60, 61, 63, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 89,90, 91, 92, 93, 94, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107, 108,110, 114, 117, 118, 119, 120, 122, 124, 125, 127, 128, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 192, 193, 195,196, 197, 198, 200, 202, 204, 205, 206, 208, 209, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 224, 226, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 242, 245, 247, 248, 251, 253, 255,256, 257, 258, 259, 260, 261, 263, 264, 265, 266, 267, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 297, 298, 300, 301, 302, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,320, 321, 323, 324, 325, 326, 327, 328, 331, 334, 335, 338, 339, 342,343, 347, 348, 349, 351, 353, 356, 357, 358, 359, 360, 361, 367, 368,369, 371, 373, 374, 375, 376, 377, 378, 379, 380, 381, 383, 385, 387,388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 401, 403, 404,405, 406, 407, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419,420, 421, 422, 425, 426, 427, 428, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446 and 447 with reference toamino acid positions set forth in SEQ ID NO:3 or 7, where correspondingamino acid positions are identified by alignment of the PH20 polypeptidewith the polypeptide set forth in SEQ ID NO:3; and provided that if themodified PH20 polypeptide contains an amino acid replacement at aposition corresponding to position 13, 47, 131, or 219 the replacementis not replacement with an Alanine (A). Among these modified PH20polypeptides are those that exhibit at least 40% of the hyaluronidaseactivity of the PH20 polypeptide not containing the amino acidreplacement, where, as in all instances herein activity is comparedunder the same conditions.

Included among these polypeptides are those that contain an amino acidreplacement in the sequence of amino acids set forth in any of SEQ IDNOs: 3, 7, 32-66, 69 and 72, or in a sequence of amino acids thatexhibits at least 91% sequence identity to any of SEQ ID NOs: 3, 7,32-66, 69, or 72. In particular, the modified PH20 polypeptide containsamino acid replacements in SEQ ID NO: 3, 7, 32-66, 69, or 72, which arepolypeptides that are a C-terminally truncated fragment of SEQ ID NO:7,or a PH20 polypeptide that has a sequence of amino acids that is atleast 91% identical to the sequence of amino acids set forth in SEQ IDNO:7. In particular, among any of such modified PH20 polypeptidesprovided herein are any including those in which the amino acidreplacement is an amino acid replacement set forth in Table 3 below. Forexample, such modified PH20 polypeptides include those that have atleast one amino acid replacement at an amino acid position correspondingto a position selected from among 1, 6, 8, 9, 10, 11, 12, 14, 15, 20,22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,46, 47, 48, 49, 50, 52, 58, 59, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75,79, 82, 83, 84, 86, 87, 89, 90, 92, 93, 94, 97, 102, 104, 107, 114, 118,120, 127, 128, 130, 131, 132, 135, 138, 139, 140, 141, 142, 143, 144,146, 147, 148, 149, 150, 151, 152, 155, 156, 158, 160, 162, 163, 164,165, 166, 167, 169, 170, 172, 173, 174, 175, 178, 179, 193, 195, 196,198, 204, 205, 206, 209, 212, 213, 215, 219, 220, 221, 222, 232, 233,234, 235, 236, 237, 238, 240, 247, 248, 249, 257, 258, 259, 260, 261,263, 267, 269, 271, 272, 273, 274, 276, 277, 278, 279, 282, 283, 285,287, 289, 291, 292, 293, 298, 305, 307, 308, 309, 310, 313, 314, 315,317, 318, 320, 321, 324, 325, 326, 328, 335, 347, 349, 351, 353, 356,359, 367, 368, 369, 371, 373, 374, 375, 376, 377, 380, 381, 383, 385,389, 392, 393, 395, 396, 399, 401, 404, 405, 406, 407, 409, 410, 412,416, 418, 419, 421, 425, 427, 428, 431, 433, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446 or 447 with reference to amino acidpositions set forth in SEQ ID NO:3. Exemplary of such replacements arethose that contain at least one amino acid replacement selected fromamong replacement with: histidine (H) at a position corresponding toposition 1; A at a position corresponding to position 1; E at a positioncorresponding to position 1; G at a position corresponding to position1; K at a position corresponding to position 1; Q at a positioncorresponding to position 1; R at a position corresponding to position1; A at a position corresponding to position 6; M at a positioncorresponding to position 8; Q at a position corresponding to position9; G at a position corresponding to position 10; H at a positioncorresponding to position 10; S at a position corresponding to position11; E at a position corresponding to position 12; I at a positioncorresponding to position 12; K at a position corresponding to position12; T at a position corresponding to position 12; V at a positioncorresponding to position 14; V at a position corresponding to position15; M at a position corresponding to position 15; S at a positioncorresponding to position 20; T at a position corresponding to position22; E at a position corresponding to position 24; H at a positioncorresponding to position 24; R at a position corresponding to position24; A at a position corresponding to position 26; E at a positioncorresponding to position 26; K at a position corresponding to position26; M at a position corresponding to position 26; Q at a positioncorresponding to position 26; R at a position corresponding to position26; D at a position corresponding to position 27; K at a positioncorresponding to position 27; Rat a position corresponding to position27; Rat a position corresponding to position 28; E at a positioncorresponding to position 29; I at a position corresponding to position29; K at a position corresponding to position 29; L at a positioncorresponding to position 29; M at a position corresponding to position29; P at a position corresponding to position 29; R at a positioncorresponding to position 29; S at a position corresponding to position29; T at a position corresponding to position 29; V at a positioncorresponding to position 29; G at a position corresponding to position30; H at a position corresponding to position 30; K at a positioncorresponding to position 30; L at a position corresponding to position30; M at a position corresponding to position 30; Rat a positioncorresponding to position 30; S at a position corresponding to position30; A at a position corresponding to position 31; C at a positioncorresponding to position 31; G at a position corresponding to position31; H at a position corresponding to position 31; I at a positioncorresponding to position 31; K at a position corresponding to position31; L at a position corresponding to position 31; P at a positioncorresponding to position 31; R at a position corresponding to position31; S at a position corresponding to position 31; Tat a positioncorresponding to position 31; V at a position corresponding to position31; W at a position corresponding to position 31; C at a positioncorresponding to position 32; F at a position corresponding to position32; G at a position corresponding to position 32; H at a positioncorresponding to position 32; W at a position corresponding to position33; G at a position corresponding to position 33; W at a positioncorresponding to position 34; Q at a position corresponding to position35; V at a position corresponding to position 35; H at a positioncorresponding to position 36; N at a position corresponding to position36; F at a position corresponding to position 37; M at a positioncorresponding to position 37; Y at a position corresponding to position38; A at a position corresponding to position 39; L at a positioncorresponding to position 39; N at a position corresponding to position39; T at a position corresponding to position 39; L at a positioncorresponding to position 40; Tat a position corresponding to position41; L at a position corresponding to position 46; R at a positioncorresponding to position 46; D at a position corresponding to position47; F at a position corresponding to position 47; T at a positioncorresponding to position 47; W at a position corresponding to position47, with F at a position corresponding to position 48; H at a positioncorresponding to position 48; K at a position corresponding to position48; N at a position corresponding to position 48; R at a positioncorresponding to position 49; D at a position corresponding to position50; S at a position corresponding to position 50; M at a positioncorresponding to position 50; N at a position corresponding to position52; Q at a position corresponding to position 52; R at a positioncorresponding to position 52; S at a position corresponding to position52; T at a position corresponding to position 52; C at a positioncorresponding to position 58; K at a position corresponding to position58; L at a position corresponding to position 58; P at a positioncorresponding to position 58; Q at a position corresponding to position58; Rat a position corresponding to position 58; H at a positioncorresponding to position 58; N at a position corresponding to position58; Y at a position corresponding to position 58; N at a positioncorresponding to position 59; K at a position corresponding to position63; L at a position corresponding to position 63; M at a positioncorresponding to position 63; Rat a position corresponding to position63; W at a position corresponding to position 63; V at a positioncorresponding to position 67; H at a position corresponding to position68; P at a position corresponding to position 68; Q at a positioncorresponding to position 68; A at a position corresponding to position69; C at a position corresponding to position 69; E at a positioncorresponding to position 69; F at a position corresponding to position69; G at a position corresponding to position 69; I at a positioncorresponding to position 69; L at a position corresponding to position69; M at a position corresponding to position 69; P at a positioncorresponding to position 69; R at a position corresponding to position69; T at a position corresponding to position 69; W at a positioncorresponding to position 69; Y at a position corresponding to position69; A at a position corresponding to position 70; C at a positioncorresponding to position 70; F at a position corresponding to position70; G at a position corresponding to position 70; H at a positioncorresponding to position 70; K at a position corresponding to position70; L at a position corresponding to position 70; N at a positioncorresponding to position 70; P at a position corresponding to position70; R at a position corresponding to position 70; S at a positioncorresponding to position 70; T at a position corresponding to position70; V at a position corresponding to position 70; Y at a positioncorresponding to position 70; G at a position corresponding to position71; N at a position corresponding to position 71; R at a positioncorresponding to position 71; S at a position corresponding to position71; K at a position corresponding to position 72; M at a positioncorresponding to position 72; Q at a position corresponding to position72; A at a position corresponding to position 73; H at a positioncorresponding to position 73; K at a position corresponding to position73; L at a position corresponding to position 73; Q at a positioncorresponding to position 73; Rat a position corresponding to position73; T at a position corresponding to position 73; W at a positioncorresponding to position 73; A at a position corresponding to position74; C at a position corresponding to position 74; E at a positioncorresponding to position 74; F at a position corresponding to position74; G at a position corresponding to position 74; H at a positioncorresponding to position 74; K at a position corresponding to position74; L at a position corresponding to position 74; M at a positioncorresponding to position 74; N at a position corresponding to position74; P at a position corresponding to position 74; R at a positioncorresponding to position 74; S at a position corresponding to position74; V at a position corresponding to position 74; W at a positioncorresponding to position 74; F at a position corresponding to position75; L at a position corresponding to position 75; M at positioncorresponding to position 75; Rat a position corresponding to position75; T at a position corresponding to position 75; L at a positioncorresponding to position 79; L at a position corresponding to position82; N at a position corresponding to position 82; V at a positioncorresponding to position 83; Q at a position corresponding to position83; S at a position corresponding to position 83; G at a positioncorresponding to position 83; E at a position corresponding to position84; F at a position corresponding to position 84; G at a positioncorresponding to position 84; N at a position corresponding to position84; R at a position corresponding to position 84; A at a positioncorresponding to position 86; H at a position corresponding to position86; K at a position corresponding to position 86; N at a positioncorresponding to position 86; S at a position corresponding to position86; T at a position corresponding to position 86; W at a positioncorresponding to position 86; C at a position corresponding to position87; G at a position corresponding to position 87; L at a positioncorresponding to position 87; M at a position corresponding to position87; R at a position corresponding to position 87; S at a positioncorresponding to position 87; T at a position corresponding to position87; V at a position corresponding to position 87; Y at a positioncorresponding to position 87; C at a position corresponding to position89; A at a position corresponding to position 90; E at a positioncorresponding to position 90; H at a position corresponding to position90; K at a position corresponding to position 90; N at a positioncorresponding to position 90; R at a position corresponding to position90; C at a position corresponding to position 92; L at a positioncorresponding to position 92; I at a position corresponding to position93; L at a position corresponding to position 93; Q at a positioncorresponding to position 93; Rat a position corresponding to position93; S at a position corresponding to position 93; T at a positioncorresponding to position 93; D at a position corresponding to position94; Q at a position corresponding to position 94; R at a positioncorresponding to position 94; A at a position corresponding to position97; C at an amino acid residue corresponding to position 97; D at aposition corresponding to position 97; E at a position corresponding toposition 97; G at a position corresponding to position 97; L at aposition corresponding to position 97; S at a position corresponding toposition 97; S at a position corresponding to position 102; T at aposition corresponding to position 102; Rat a position corresponding toposition 104; L at a position corresponding to position 107; A at aposition corresponding to position 114; Q at a position corresponding toposition 118; H at a position corresponding to position 120; F at aposition corresponding to position 120; I at a position corresponding toposition 120; S at a position corresponding to position 120; V at aposition corresponding to position 120; Y at a position corresponding toposition 120; E at a position corresponding to position 127; H at aposition corresponding to position 127; N at a position corresponding toposition 127; Q at a position corresponding to position 127; Rat aposition corresponding to position 127; I at a position corresponding toposition 128; Rat a position corresponding to position 130; G at aposition corresponding to position 131; I at a position corresponding toposition 131; M at a position corresponding to position 131; Q at aposition corresponding to position 131; Rat a position corresponding toposition 131; V at a position corresponding to position 131; N at aposition corresponding to position 132; L at a position corresponding toposition 132; D at a position corresponding to position 135; G at aposition corresponding to position 135; Rat a position corresponding toposition 135, with L at a position corresponding to position 138; T at aposition corresponding to position 139; K at a position corresponding toposition 140; H at a position corresponding to position 141; Rat aposition corresponding to position 141; S at a position corresponding toposition 141; W at a position corresponding to position 141; Y at aposition corresponding to position 141; D at a position corresponding toposition 142; G at a position corresponding to position 142; K at aposition corresponding to position 142; N at a position corresponding toposition 142; P at a position corresponding to position 142; Q at aposition corresponding to position 142; R at a position corresponding toposition 142; S at a position corresponding to position 142; T at aposition corresponding to position 142; G at a position corresponding toposition 143; K at a position corresponding to position 143; Rat aposition corresponding to position 144; T at a position corresponding toposition 144; P at a position corresponding to position 146; R at aposition corresponding to position 146; A at a position corresponding toposition 147; F at a position corresponding to position 147; L at aposition corresponding to position 147; R at a position corresponding toposition 147; S at a position corresponding to position 147; V at aposition corresponding to position 147; H at a position corresponding toposition 148; K at a position corresponding to position 148; Q at aposition corresponding to position 148; T at a position corresponding toposition 149; V at a position corresponding to position 149; A at aposition corresponding to position 150; D at a position corresponding toposition 150; G at a position corresponding to position 150; N at aposition corresponding to position 150; S at a position corresponding toposition 150; W at a position corresponding to position 150; Y at aposition corresponding to position 150; A at a position corresponding toposition 151; H at a position corresponding to position 151; K at aposition corresponding to position 151; L at a position corresponding toposition 151; M at a position corresponding to position 151; Q at aposition corresponding to position 151; Rat a position corresponding toposition 151; S at a position corresponding to position 151; Tat aposition corresponding to position 151; V at a position corresponding toposition 151; W at a position corresponding to position 151; Y at aposition corresponding to position 151; Rat a position corresponding toposition 152; T at a position corresponding to position 152; W at aposition corresponding to position 152; D at a position corresponding toposition 155; G at a position corresponding to position 155; K at aposition corresponding to position 155; Rat a position corresponding toposition 155; D at a position corresponding to position 156; Q at aposition corresponding to position 158; S at a position corresponding toposition 158; S at a position corresponding to position 160; E at aposition corresponding to position 162; A at a position corresponding toposition 163; E at a position corresponding to position 163; K at aposition corresponding to position 163; Q at a position corresponding toposition 163; Rat a position corresponding to position 163; S at aposition corresponding to position 163; M at a position corresponding toposition 164; V at a position corresponding to position 164; D at aposition corresponding to position 165; F at a position corresponding toposition 165; N at a position corresponding to position 165; S at aposition corresponding to position 165; V at a position corresponding toposition 165; A at a position corresponding to position 166; E at aposition corresponding to position 166; F at a position corresponding toposition 166; H at a position corresponding to position 166; L at aposition corresponding to position 166; Q at a position corresponding toposition 166; Rat a position corresponding to position 166; Tat aposition corresponding to position 166; W at a position corresponding toposition 166; Y at a position corresponding to position 166; D at aposition corresponding to position 167; L at a position corresponding toposition 169; Rat a position corresponding to position 170; A at aposition corresponding to position 172; Rat a position corresponding toposition 173; G at a position corresponding to position 174; K at aposition corresponding to position 174; N at a position corresponding toposition 174; Rat a position corresponding to position 174; T at aposition corresponding to position 174; T at a position corresponding toposition 175; K at a position corresponding to position 178; Rat aposition corresponding to position 178; K at a position corresponding toposition 179; Q at a position corresponding to position 193; T at aposition corresponding to position 195; N at a position corresponding toposition 195; with E at a position corresponding to position 196; Rat aposition corresponding to position 196; with D at a positioncorresponding to position 198; P at a position corresponding to position204; A at a position corresponding to position 205; E at a positioncorresponding to position 205; L at a position corresponding to position205; T at a position corresponding to position 205; I at a positioncorresponding to position 206; K at a position corresponding to position206; L at a position corresponding to position 206; R at a positioncorresponding to position 206; R at a position corresponding to position209; N at a position corresponding to position 212; S at a positioncorresponding to position 212; A at a position corresponding to position213; M at a position corresponding to position 213; N at a positioncorresponding to position 213; H at a position corresponding to position215; M at a position corresponding to position 215; I at a positioncorresponding to position 219; K at a position corresponding to position219; S at a position corresponding to position 219; H at a positioncorresponding to position 220; I at a position corresponding to position220; L at a position corresponding to position 220; V at a positioncorresponding to position 220; Q at a position corresponding to position221; G at a position corresponding to position 222; F at a positioncorresponding to position 232; G at a position corresponding to position233; K at a position corresponding to position 233; Rat a positioncorresponding to position 233; M at a position corresponding to position234; A at a position corresponding to position 235; R at a positioncorresponding to position 236; C at a position corresponding to position237; E at a position corresponding to position 237; H at a positioncorresponding to position 237; Q at a position corresponding to position237; T at a position corresponding to position 237; E at a positioncorresponding to position 238; H at a position corresponding to aminoacid position 238; S at a position corresponding to position 238; A at aposition corresponding to position 240; Q at a position corresponding toposition 240; I at a position corresponding to position 247; A at aposition corresponding to position 248; V at a position corresponding toposition 249; G at a position corresponding to position 257; T at aposition corresponding to position 257; Rat a position corresponding toposition 257; N at a position corresponding to position 258; S at aposition corresponding to position 258; P at a position corresponding toposition 259; M at a position corresponding to position 260; Y at aposition corresponding to position 260; A at a position corresponding toposition 261; K at a position corresponding to position 261; N at aposition corresponding to position 261; K at a position corresponding toposition 263; Rat a position corresponding to position 263; T at aposition corresponding to position 267; A at a position corresponding toposition 269; L at a position corresponding to position 271; M at aposition corresponding to position 271; D at a position corresponding toposition 272; Tat a position corresponding to position 272; H at aposition corresponding to position 273; Y at a position corresponding toposition 273; F at a position corresponding to position 274; D at aposition corresponding to position 276; H at a position corresponding toposition 276; M at a position corresponding to position 276; R at aposition corresponding to position 276; S at a position corresponding toposition 276; Y at a position corresponding to position 276; A at aposition corresponding to position 277; E at a position corresponding toposition 277; H at a position corresponding to position 277; K at aposition corresponding to position 277; M at a position corresponding toposition 277; N at a position corresponding to position 277; Q at aposition corresponding to position 277; R at a position corresponding toposition 277; S at a position corresponding to position 277; T at aposition corresponding to position 277; E at a position corresponding toposition 278; F at a position corresponding to position 278; G at aposition corresponding to position 278; H at a position corresponding toposition 278; K at a position corresponding to position 278; N at aposition corresponding to position 278; Rat a position corresponding toposition 278; S at a position corresponding to position 278; T at aposition corresponding to position 278; Y at a position corresponding toposition 278; H at a position corresponding to position 279; M at aposition corresponding to position 282; S at a position corresponding toposition 283; H at a position corresponding to position 285; T at aposition corresponding to position 287; S at a position corresponding toposition 289; S at a position corresponding to position 291; V at aposition corresponding to position 291; C at a position corresponding toposition 292; F at a position corresponding to position 292; H at aposition corresponding to position 292; K at a position corresponding toposition 292; R at a position corresponding to position 292; V at aposition corresponding to position 292; A at a position corresponding toposition 293; C at a position corresponding to position 293; D at aposition corresponding to position 293; F at a position corresponding toposition 293; K at a position corresponding to position 293; M at aposition corresponding to position 293; P at a position corresponding toposition 293; Q at a position corresponding to position 293; V at aposition corresponding to position 293; Y at a position corresponding toposition 293; G at a position corresponding to position 298; E at aposition corresponding to position 305; G at a position corresponding toposition 307; D at a position corresponding to position 308; G at aposition corresponding to position 308; K at a position corresponding toposition 308; N at a position corresponding to position 308; Rat aposition corresponding to position 308; E at a position corresponding toposition 309; G at a position corresponding to position 309; H at aposition corresponding to position 309; L at a position corresponding toposition 309; M at a position corresponding to position 309; N at aposition corresponding to position 309; Q at a position corresponding toposition 309; Rat a position corresponding to position 309; S at aposition corresponding to position 309; T at a position corresponding toposition 309; V at a position corresponding to position 309; A at aposition corresponding to position 310; G at a position corresponding toposition 310; Q at a position corresponding to position 310; S at aposition corresponding to position 310; A at a position corresponding toposition 313; G at a position corresponding to position 313; H at aposition corresponding to position 313; K at a position corresponding toposition 313; P at a position corresponding to position 313; Rat aposition corresponding to position 313; Tat a position corresponding toposition 313; Y at a position corresponding to position 313; with S at aposition corresponding to position 314; Y at a position corresponding toposition 314; A at a position corresponding to position 315; H at aposition corresponding to position 315; Y at a position corresponding toposition 315; A at a position corresponding to position 317; I at aposition corresponding to position 317; K at a position corresponding toposition 317; N at a position corresponding to position 317; Q at aposition corresponding to position 317; Rat a position corresponding toposition 317; S at a position corresponding to position 317; T at aposition corresponding to position 317; W at a position corresponding toposition 317; D at a position corresponding to position 318; H at aposition corresponding to position 318; K at a position corresponding toposition 318; M at a position corresponding to position 318; R at aposition corresponding to position 318; H at a position corresponding toposition 320; K at a position corresponding to position 320; R at aposition corresponding to position 320; Rat a position corresponding toposition 321; S at a position corresponding to position 321; N at aposition corresponding to position 324; Rat a position corresponding toposition 324; A at a position corresponding to position 325; D at aposition corresponding to position 325; E at a position corresponding toposition 325; G at a position corresponding to position 325; H at aposition corresponding to position 325; K at a position corresponding toposition 325; M at a position corresponding to position 325; N at aposition corresponding to position 325; Q at a position corresponding toposition 325; S at a position corresponding to position 325; V at aposition corresponding to position 325; L at a position corresponding toposition 326; V at a position corresponding to position 326; C at aposition corresponding to position 328; G at a position corresponding toposition 328; I at a position corresponding to position 328; K at aposition corresponding to position 328; L at a position corresponding toposition 328; S at a position corresponding to position 328; Y at aposition corresponding to position 328; S at a position corresponding toposition 335; A at a position corresponding to position 347; G at aposition corresponding to position 347; S at a position corresponding toposition 347; M at a position corresponding to position 349; R at aposition corresponding to position 349; S at a position corresponding toposition 351; V at a position corresponding to position 353; with H at aposition corresponding to position 356; S at a position corresponding toposition 356; E at a position corresponding to position 359; H at aposition corresponding to position 359; T at a position corresponding toposition 359; A at a position corresponding to position 367; G at aposition corresponding to position 367; K at a position corresponding toposition 367; S at a position corresponding to position 367; A at aposition corresponding to position 368; E at a position corresponding toposition 368; K at a position corresponding to position 368; L at aposition corresponding to amino acid position 368; M at a positioncorresponding to amino acid position 368; R at a position correspondingto position 368; T at a position corresponding to amino acid position368; H at a position corresponding to position 369; R at a positioncorresponding to position 369; F at a position corresponding to position371; H at a position corresponding to position 371; K at a positioncorresponding to position 371; L at a position corresponding to position371; Rat a position corresponding to position 371; S at a positioncorresponding to position 371; M at a position corresponding to position373; H at a position corresponding to position 374; P at a positioncorresponding to position 374; A at a position corresponding to position375; G at a position corresponding to position 375; K at a positioncorresponding to position 375; Rat a position corresponding to position375; D at a position corresponding to position 376; E at a positioncorresponding to position 376; Q at a position corresponding to position376; Rat a position corresponding to position 376; T at a positioncorresponding to position 376; V at a position corresponding to position376; Y at a position corresponding to position 376; D at a positioncorresponding to position 377; E at a position corresponding to position377; H at a position corresponding to position 377; K at a positioncorresponding to position 377; P at a position corresponding to position377; Rat a position corresponding to position 377; S at a positioncorresponding to position 377; T at a position corresponding to position377; W at a position corresponding to position 380; Y at a positioncorresponding to position 380; S at a position corresponding to position381; I at a position corresponding to position 383; K at a positioncorresponding to position 383; L at a position corresponding to position383; S at a position corresponding to position 383; A at a positioncorresponding to position 385; Q at a position corresponding to position385; V at a position corresponding to position 385; A at a positioncorresponding to position 389; G at a position corresponding to position389; L at a position corresponding to position 389; K at a positioncorresponding to position 389; Q at a position corresponding to position389; S at a position corresponding to position 389; A at a positioncorresponding to position 392; F at a position corresponding to position392; M at a position corresponding to position 392; Q at a positioncorresponding to position 392; R at a position corresponding to position392; V at a position corresponding to position 392; F at a positioncorresponding to position 393; M at a position corresponding to position393; A at a position corresponding to position 395; H at a positioncorresponding to position 395; Rat a position corresponding to position395; A at a position corresponding to position 396; H at a positioncorresponding to position 396; Q at a position corresponding to position396; S at a position corresponding to position 396; K at a positioncorresponding to position 399; M at a position corresponding to position399; T at a position corresponding to position 399; V at a positioncorresponding to position 399; W at a position corresponding to position399; A at a position corresponding to position 401; E at a positioncorresponding to position 401; A at a position corresponding to position404; G at a position corresponding to position 405; F at a positioncorresponding to position 406; N at a position corresponding to position406; A at a position corresponding to position 407; D at a positioncorresponding to position 407; E at a position corresponding to position407; F at a position corresponding to position 407; H at a positioncorresponding to position 407; Q at a position corresponding to position407; P at a position corresponding to position 407; A at a positioncorresponding to position 409; Q at a position corresponding to position409; Tat a position corresponding to position 410; Q at a positioncorresponding to position 412; Rat a position corresponding to position412; V at a position corresponding to position 412; L at a positioncorresponding to position 416; E at a position corresponding to position418; L at a position corresponding to position 418; P at a positioncorresponding to position 418; Rat a position corresponding to position418; V at a position corresponding to position 418; F at a positioncorresponding to position 419; H at a position corresponding to position419; I at a position corresponding to position 419; K at a positioncorresponding to position 419; Rat a position corresponding to position419; S at a position corresponding to position 419; Y at a positioncorresponding to position 419; A at a position corresponding to position421; H at a position corresponding to position 421; K at a positioncorresponding to position 421; N at a position corresponding to position421; Q at a position corresponding to position 421; R at a positioncorresponding to position 421; S at a position corresponding to position421; G at a position corresponding to position 425; K at a positioncorresponding to position 425; Q at a position corresponding to position427; T at a position corresponding to position 427; L at a positioncorresponding to position 428; A at a position corresponding to position431; G at a position corresponding to position 431; E at a positioncorresponding to position 431; H at a position corresponding to position431; K at a position corresponding to position 431; L at a positioncorresponding to position 431; N at a position corresponding to position431; Q at a position corresponding to position 431; Rat a positioncorresponding to position 431; S at a position corresponding to position431; V at a position corresponding to position 431; A at a positioncorresponding to position 433; H at a position corresponding to position433; I at a position corresponding to position 433; K at a positioncorresponding to position 433; L at a position corresponding to position433; Rat a position corresponding to position 433; T at a positioncorresponding to position 433; V at a position corresponding to position433; W at a position corresponding to position 433; K at a positioncorresponding to position 436; I at a position corresponding to position437; M at a position corresponding to position 437; A at a positioncorresponding to position 438; D at a position corresponding to position438; Eat a position corresponding to position 438; L at a positioncorresponding to position 438; N at a position corresponding to position438; T at a position corresponding to position 438; A at a positioncorresponding to position 439; C at a position corresponding to position439; K at a position corresponding to position 439; P at a positioncorresponding to position 439; Q at a position corresponding to position439; T at a position corresponding to position 439; V at a positioncorresponding to position 439; D at a position corresponding to position440; H at a position corresponding to position 440; M at a positioncorresponding to position 440; P at a position corresponding to position440; R at a position corresponding to position 440; S at a positioncorresponding to position 440; A at a position corresponding to position441; F at a position corresponding to position 441; C at a positioncorresponding to position 442; G at a position corresponding to position442; R at a position corresponding to position 442; A at a positioncorresponding to position 443; E at a position corresponding to position443; F at a position corresponding to position 443; G at a positioncorresponding to position 443; M at a position corresponding to position443; N at a position corresponding to position 443; E at a positioncorresponding to position 444; H at a position corresponding to position444; V at a position corresponding to position 444; H at a positioncorresponding to position 445; M at a position corresponding to position445; N at a position corresponding to position 445; P at a positioncorresponding to position 445; Q at a position corresponding to position445; S at a position corresponding to position 445; T at a positioncorresponding to position 445; V at a position corresponding to position445; W at a position corresponding to position 445; A at a positioncorresponding to position 446; M at a position corresponding to position446; W at a position corresponding to position 446; D at a positioncorresponding to position 447; E at a position corresponding to position447; G at a position corresponding to position 447; I at a positioncorresponding to position 447; N at a position corresponding to position447; P at a position corresponding to position 447; Q at a positioncorresponding to position 447; T at a position corresponding to position447, and/or replacement with V at a position corresponding to position447, each with reference to amino acid positions set forth in SEQ IDNO:3. Among these modified PH20 polypeptides are those that exhibit atleast 40% of the activity of the PH20 that does not contain theparticular amino acid replacement. Activity can vary between, forexample, 40% to 5000%, 40% to 2000%, 40% to 1000%, 40% to 500%, 40% to100%, 80% to 2000%, 80% to 600%, 80% to 200%, 80% to 300%, of thehyaluronidase activity of the PH20 polypeptide not containing the aminoacid replacement. Such activity includes at least 50%, 60%, 70%, 80%,90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000% or more of thehyaluronidase activity of the PH20 polypeptide not containing the aminoacid replacement, where, as in all instances herein, the activities arecompared under the same conditions.

In particular, provided are modified PH20 polypeptides that contain atleast one amino acid replacement in a PH20 polypeptide set forth in SEQID NO:7, a C-terminally truncated fragment thereof, or in a PH20polypeptide that has a sequence of amino acids that is at least 91%identical to the sequence of amino acids set forth in SEQ ID NO:7 or acorresponding truncated fragment, where: the modified PH20 polypeptidesexhibit less than 20% of the hyaluronidase activity of the PH20polypeptide not containing the amino acid replacement, where activitiesare compared under the same conditions; the amino acid replacement(s) isat an amino acid position corresponding to a position selected fromamong 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 25, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 143, 144, 145, 149,150, 152, 153, 154, 155, 156, 157, 158, 159, 161, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 331,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 408, 410, 411, 412, 413, 414, 415, 416, 417, 419,420, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 437,438, 439, 440, 441, 442, 443, 444, or 447 with reference to amino acidpositions set forth in SEQ ID NO:3 or 7;

corresponding amino acid positions are identified by alignment of thePH20 polypeptide with the polypeptide set forth in SEQ ID NO:3; andprovided that:

(i) if the modified PH20 polypeptide contains an amino acid replacementat a position corresponding to position 200, 333, 358 or 393 thereplacement is not replacement with an Alanine (A).

(ii) if the modified PH20 polypeptide contains an amino acid replacementat a position corresponding to position 111 or 249 the replacement isnot replacement with an asparagine (N);

(iii) if the modified PH20 polypeptide contains an amino acidreplacement at a position corresponding to position 113 the replacementis not replacement with a glutamine (Q);

(iv) if the modified PH20 polypeptide contains an amino acid replacementat a position corresponding to position 176 the replacement is notreplacement with a glycine (G); and

(v) if the modified PH20 polypeptide contains an amino acid replacementat a position corresponding to position 252 the replacement is notreplacement with a threonine (T).

Exemplary of such modified PH20 polypeptides are any that contain aminoacid replacement(s) in a PH20 polypeptide that has the sequence of aminoacids set forth in any of SEQ ID NOs: 3, 7, 32-66, 69, or 72, or in asequence of amino acids that exhibits at least 91% sequence identity toany of SEQ ID NOs: 3, 7, 32-66, 69, or 72. For example, the modifiedPH20 polypeptide contains amino acid replacement(s) in SEQ ID NOs: 3, 7,32-66, 69, or 72, which are polypeptides that are a C-terminallytruncated fragment of SEQ ID NO:7, or a PH20 polypeptide that has asequence of amino acids that is at least 91% identical to the sequenceof amino acids set forth in SEQ ID NO:7. In examples of such modifiedPH20 polypeptides provided herein, the modified PH20 polypeptides canexhibit similar or the same activity as the PH20 without themodification, or can exhibit increased activity or activity that is lessthan 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% or less of the hyaluronidaseactivity of the PH20 polypeptide not containing the amino acidreplacement. Exemplary of such modified PH20 polypeptides are any setforth in Table 5.

Among any and all of the modified PH20 polypeptides provided herein andabove, the modified PH20 polypeptide is one that does not consist of thesequence of amino acids set forth in any of SEQ ID NOs: 3, 6-66, 69-72,856-861, 869 or 870. In particular, among any of the modified PH20polypeptides provided herein above or elsewhere herein are any thatcontain an amino acid replacement(s) in a PH20 polypeptide having thesequence of amino acids set forth any of SEQ ID NO: 3, 7, 69 or 72provided that: (i) where the modified PH20 polypeptide includes only asingle amino acid replacement the replacement does not corresponds toamino acid replacements V12A, N47A, D111N, E113Q, N131A, R176G, N200A,N219A, E249Q, R252T, N333A or N358A, with reference to amino acidpositions set forth in SEQ ID NO:3; (ii) where the modified PH20polypeptide includes only two amino acid replacements the replacementsdo not correspond to amino acid replacements P 13A/L464W, N47A/N131A,N47A/N219A, N131A/N219A or N333A/N358A with reference to positions setforth in SEQ ID NO:3; and (iii) where the modified PH20 polypeptideincludes only three amino acid replacements the replacements do notcorrespond to amino acid replacements N47A/N131A/N219A, with referenceto amino acid positions set forth in SEQ ID NO:3.

Any of the above modified PH20 polypeptides and any provided herein anddescribed above and below can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, or more of the amino acid replacements. Themodified PH20 polypeptides can include a signal sequence, including thenative sequence or a heterologous sequence or a modified sequence, andalso include a mature PH20 polypeptide that lacks the signal sequence.

Among any of the modified PH20 polypeptides provided herein above ordescribed below are modified PH20 polypeptides that contain or have thesequence of amino acids set forth in any of SEQ ID NOs: 73-855 or asequence of amino acids that exhibits at least 75%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to asequence of amino acids set forth in any of SEQ ID NOs: 73-855 and thatcontains at least one amino acid replacement, such as any describedabove or elsewhere herein, with reference to positions compared to thesequence of amino acids set forth in SEQ ID NO:3. In any of the examplesof the modified PH20 polypeptides provided herein, the modified PH20polypeptide does not have or contain the sequence of amino acids setforth in any of SEQ ID NOs: 8-31, 69-72, 856-861, 869 or 870.

The modified PH20 polypeptides provided herein can be substantiallypurified or isolated, can exhibit catalytic activity at neutral pH, canbe secreted upon expression from cells and are soluble in thesupernatant, and/or can include modified amino acids, such as amodification selected from among glycosylation, sialation, albumination,farnysylation, carboxylation, hydroxylation, conjugation to a polymer,such as PEGylation or conjugation to dextran, conjugation to anothermoiety, such as a multimerization domain, toxin, detectable label ordrug, and phosphorylation. The modified PH20 polypeptide can beglycosylated, such as by containing at least an N-acetylglucosaminemoiety linked to each of at least three asparagine (N) residues, where,for example, the three asparagine residues correspond to amino acidresidues 200, 333 and 358 of SEQ ID NO:3. Multimerization domainsinclude Fc domains.

Also provided are nucleic acid molecules that encode any of the modifiedPH20 polypeptides provided herein. Vectors, eukaryotic and prokaryotic,that contain the nucleic acid molecules are provided. The vectorsinclude expression vectors and include mammalian vectors, includingviral vectors. Viral vectors include adenovirus vectors, retrovirusvectors, vaccinia virus vectors, herpes simplex virus andcytomegalovirus vectors, and other such viral vectors. Of interest areoncolytic vectors that accumulate in or are targeted to tumors. Alsoprovided are cells that contain the nucleic acid molecules and cellsthat contain the vectors. The cells can be prokaryotic or eukaryotic,particularly mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

Also provided herein is a modified PH20 polypeptide that is produced byany of the provided cells. Thus, provided herein are methods ofproducing a modified PH20 polypeptide by culturing any of the cellsprovided herein under conditions whereby an encoded modified PH20polypeptide is produced and secreted by the cell, and recovering theexpressed polypeptide. Also provided herein is a method of producing amodified PH20 polypeptide by introducing any of the nucleic acidsprovided herein or any of the vectors provided herein into a cellcapable of incorporating N-linked sugar moieties into the polypeptide,culturing the cell under conditions whereby an encoded modified PH20polypeptide is produced and secreted by the cell, and recovering theexpressed polypeptide. In such examples, the nucleic acid is operablylinked to a promoter. The cultured cell can be a eukaryotic cell, suchas a mammalian cell, for example, a Chinese hamster ovary (CHO) cell.

Also provided are pharmaceutical compositions that contain any of themodified PH20 polypeptides provided herein or any of the nucleic acidsor vectors provided herein. The compositions can be formulated withother agents and/or with other components, such as preservatives. Thecompositions can be formulated so that the components, particularly thePH20 and any other active agent, remain active or are stable underpreselected conditions. In addition, as described herein, the PH20polypeptides are modified so that they exhibit increased stability undervarious conditions. For example, provided are compositions in which themodified PH20 polypeptide is stable (i.e., retains activity as describedherein) at a temperature from or from about 2° C. to 8° C., inclusive,for at least 1 month or is stable at a temperature from or from about30° C. to 42° C., inclusive, for at least 3 days. Provided arecompositions in which the modified PH20 polypeptide in the compositionis stable at a temperature from or from about 2° C. to 8° C., inclusive,for at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months,at least 8 months, at least 9 months, at least 10 months, at least 11months, at least 12 months, 13 months, 14 months, 15 months, 16 months,17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 monthsor 30 months. Also provided are compositions in which the modified PH20polypeptide in the composition is stable at a temperature from or fromabout 30° C. to 42° C., inclusive, for at least 3 days, at least 4 days,5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45days, 50 days, 60 days or more. The pharmaceutical compositions cancontain a pharmaceutically acceptable excipient.

The conditions, formulations, components, and modified PH20 polypeptideare chosen to achieve a desired stability. The pharmaceuticalcompositions can be formulated for direct administration or can requiredilution. They can be formulated for multiple or single dosageadministration. Exemplary compositions include concentrations ofmodified PH20 between or about between 0.1 μg/mL to 100 μg/mL, 1 μg/mLto 50 μg/mL or 1 μg/mL to 20 μg/mL, or 10 U/mL to 5000 U/mL, 50 U/mL to4000 U/mL, 100 U/mL to 2000 U/mL, 300 U/mL to 2000 U/mL, 600 U/mL to2000 U/mL, or 100 U/mL to 1000 U/mL. Exemplary salts include NaCl at aconcentration, for example, of less than or about or 200 mM, 180 mM, 150mM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM,50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, orbetween or about between 0.1 mM to 200 mM, 0.1 mM to 100 mM, 120 mM to200 mM, 10 mM to 50 mM, 10 mM to 90 mM, 80 mM to 200 mM, 80 mM to 140mM, 50 mM to 100 mM, 80 mM to 100 mM, 50 mM to 80 mM, 100 mM to 140 mMor 120 mM to 140 mM.

The pharmaceutical compositions can contain an anti-microbiallyeffective amount of a preservative or mixture of preservatives, such asone, two, three, four or more of a phenolic preservative(s), anon-phenolic preservative(s) or a phenolic preservative(s) and anon-phenolic preservative(s), such as, but not limited to, phenol,m-cresol, methylparaben, benzyl alcohol, thimerosal, benzalkoniumchloride, 4-chloro-1-butanol, chlorhexidine dihydrochloride,chlorhexidine digluconate, L-phenylalanine, EDTA, bronopol,phenylmercuric acetate, glycerol, imidurea, chlorhexidine, sodiumdehydroacetate, o-cresol, p-cresol, chlorocresol, cetrimide,benzethonium chloride, ethyl paraben, propylparaben, butylparaben andany combinations thereof. Phenols include, for example, phenol,metacresol (m-cresol), benzyl alcohol, and parabens, such asmethylparaben or propylparaben. Anti-microbial effective concentrationsof one or more preservative agents (as a percentage (%) of massconcentration (w/v)) can be between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or0.3% to 0.4% inclusive. Examples thereof are pharmaceutical compositionswhere the preservatives are phenol, m-cresol or phenol and m-cresol andthe amount as a % of mass concentration (w/v) in the formulation isbetween or about between 0.1% to 0.25% phenol and between or aboutbetween 0.05% to 0.2% m-cresol, is between or about between 0.10% to0.2% phenol and between or about between 0.6% to 01.8% m-cresol, betweenor about between 0.1% to 0.15% phenol and 0.8% to 0.15% m-cresol, isbetween or about between 0.10% to 0.15% phenol and between or aboutbetween 0.06 to 0.09% m-cresol or is between or about between 0.12% to0.18% phenol and between or about between 0.14 to 0.22% m-cresol.

The pharmaceutical compositions can contain a further therapeuticallyactive agent. The active agent can be formulated in the composition orprovided as a combination with the PH20-containing composition, but in aseparate composition for administration separately, sequentially,intermittently, simultaneously or together. Therapeutically activeagents include, for example, an agent selected from among achemotherapeutic agent, an analgesic agent, an anti-inflammatory agent,an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent,an anti-Parkinson agent, an anti-malarial agent, an anticonvulsantagent, an anti-depressant agent, and antiarthritics agent, ananti-fungal agent, an antihypertensive agent, an antipyretic agent, ananti-parasite agent, an antihistamine agent, an alpha-adrenergic agonistagent, an alpha blocker agent, an anesthetic agent, a bronchial dilatoragent, a biocide agent, a bactericide agent, a bacteriostat agent, abeta adrenergic blocker agent, a calcium channel blocker agent, acardiovascular drug agent, a contraceptive agent, a decongestant agent,a diuretic agent, a depressant agent, a diagnostic agent, a electrolyteagent, a hypnotic agent, a hormone agent, a hyperglycemic agent, amuscle relaxant agent, a muscle contractant agent, an ophthalmic agent,a parasympathomimetic agent, a psychic energizer agent, a sedativeagent, a sympathomimetic agent, a tranquilizer agent, an urinary agent,a vaginal agent, a viricide agent, a vitamin agent, a non-steroidalanti-inflammatory agent, an angiotensin converting enzyme inhibitoragent, a polypeptide, a protein, a nucleic acid, a drug, an organicmolecule and a sleep inducer. Exemplary of such agents are antibodies,particularly monoclonal antibodies, an Immune Globulin preparation, abisphosphonate, a cytokine, a chemotherapeutic agent, a coagulationfactor and an insulin. Insulins include, for example, basal insulins andfast-acting insulin, such as regular insulin, particularly recombinanthuman insulin, and insulin analogs, such as insulin lispro, insulinaspart or insulin glulisine. Particular fast-acting insulins are thosewith an A chain having a sequence of amino acids set forth in SEQ IDNO:862 and a B chain having a sequence of amino acids set forth in SEQID NO:863 or an insulin with an A chain with a sequence of amino acidsset forth as amino acid residue positions 88-108 of SEQ ID NO:864 and aB chain with a sequence of amino acids set forth as amino acid residuepositions 25-54 of SEQ ID NO:864 or an insulin analog that is selectedfrom among an insulin having an A chain with a sequence of amino acidsset forth in SEQ ID NO:862 and a B chain having a sequence of aminoacids set forth in any of SEQ NOs:865-867. The amount of fast-actinginsulin in the compositions can be empirically determined, but typicallycan be 10 U/mL to 1000 U/mL, 50 U/mL to 500 U/mL, 100 U/mL to 1000 U/mLor 500 U/mL to 1000 U/mL, inclusive.

In particular examples, provided herein is a pharmaceutical compositioncontaining any of the modified PH20 polypeptides provided herein thatexhibit increased stability to a phenolic preservative and an insulin,such as a fast-acting insulin. The modified PH20 polypeptides andinsulin can be provided in therapeutically effective amounts. Forexample, provided herein is a pharmaceutical composition that containsany of the modified PH20 polypeptides provided herein that exhibitsincreased stability to a phenolic preservative in an amount that is fromor from about 100 U/mL to 1000 U/mL and a fast-acting insulin in anamount that is from or from about 10 U/mL to 1000 U/mL. For example, thefast-acting insulin can be an insulin analog, such as insulin lispro,insulin aspart or insulin glulisine or other analog. Any of suchpharmaceutical compositions can be formulated at a pH that is from orfrom about 7.0 to 7.6. Any of such pharmaceutical compositions also canbe formulated to contain salt, such as NaCl, at a concentration that isfrom or from about 0.1 mM to 200 mM and/or an anti-microbial effectiveamount of at least one preservative where the composition generallycontains at least one phenolic preservative. The anti-microbialeffective amount is a total amount of one or more preservative agents asa percentage (%) of mass concentration (w/v) that is or is between 0.05%and 0.6%. The phenolic preservative(s) can be a phenol, metacresol(m-cresol), benzyl alcohol, or a paraben. In any of the above examplesof a pharmaceutical composition, the composition also can contain asurfactant, such as a polypropylene glycol, polyethylene glycol,glycerin, sorbitol, poloxamer or polysorbate, in an amount as a % ofmass concentration (w/v) in the formulation that is at least or at leastabout 0.001%; a buffering agent that is a non-metal binding agent or isa metal binding agent, such as Tris, histidine, phosphate or citrate,wherein the concentration of the buffering agent is between or betweenabout 1 mM to 100 mM; glycerin in a concentration less than 60 mM; anantioxidant, such as cysteine, tryptophan or methionine, at aconcentration between or from about between 2 mM to 50 mM, inclusive;and/or zinc at a concentration of between or about between 0.001 to 0.1mg per 100 units of insulin (mg/100U). Also provided herein are closedloop systems, insulin pumps including continuous subcutaneous infusioninsulin (CSII) pumps and insulin pens that contain any of thepharmaceutical compositions. The pharmaceutical compositions can be usedin methods or uses for treating diabetes, such as type 1 diabetesmellitus, type 2 diabetes mellitus or gestational diabetes.

Other therapeutic agents in any of the pharmaceutical compositionsprovided herein include, but are not limited to Adalimumabs, AgalsidaseBetas, Alefacepts, Ampicillins, Anakinras, Antipoliomyelitic Vaccines,Anti-Thymocytes, Azithromycins, Becaplermins, Caspofungins, Cefazolins,Cefepimes, Cefotetans, Ceftazidimes, Ceftriaxones, Cetuximabs,Cilastatins, Clavulanic Acids, Clindamycins, Darbepoetin Alfas,Daclizumabs, Diphtheria, Diphtheria antitoxins, Diphtheria Toxoids,Efalizumabs, Epinephrines, Erythropoietin Alphas, Etanercepts,Filgrastims, Fluconazoles, Follicle-Stimulating Hormones, FollitropinAlphas, Follitropin Betas, Fosphenytoins, Gadodiamides, Gadopentetates,Gatifloxacins, Glatiramers, GM-CSF's, Goserelins, Goserelin acetates,Granisetrons, Haemophilus Influenza B's, Haloperidols, Hepatitisvaccines, Hepatitis A Vaccines, Hepatitis B Vaccines, IbritumomabTiuxetans, Ibritumomabs, Tiuxetans, Immunoglobulins, Hemophilusinfluenza vaccines, Influenza Virus Vaccines, Infliximabs, Insulinlispro, 75% neutral protamine lispro (NPL)/25% insulin lispro, 50%neutral protamine Hagedorn (NPH)/50% regular insulin, 70% NPH/30%regular insulin; Regular insulin, NPH insulin, Ultra insulin, Ultralenteinsulin, and Insulin Glargines, Interferons, Interferon alpha,Interferon Betas, Interferon Gammas, Interferon alpha-2a, Interferonalpha 2-b, Interferon Alphacon, Interferon alpha-n, Interferon Betas,Interferon Beta-1a's, Interferon Gammas, Interferon alpha-con,Iodixanols, Iohexols, Iopamidols, Ioversols, Ketorolacs, Laronidases,Levofloxacins, Lidocaines, Linezolids, Lorazepams, Measles Vaccines,Measles virus, Mumps viruses, Measles-Mumps-Rubella Virus Vaccines,Rubella vaccines, Medroxyprogesterones, Meropenems, Methylprednisolones,Midazolams, Morphines, Octreotides, Omalizumabs, Ondansetrons,Palivizumabs, Pantoprazoles, Pegaspargases, Pegfilgrastims,Peg-Interferon Alpha-2a's, Peg-Interferon Alpha-2b's, Pegvisomants,Pertussis vaccines, Piperacillins, Pneumococcal Vaccines andPneumococcal Conjugate Vaccines, Promethazines, Reteplases, Somatropins,Sulbactams, Sumatriptans, Tazobactams, Tenecteplases, Tetanus PurifiedToxoids, Ticarcillins, Tositumomabs, Triamcinolones, TriamcinoloneAcetonides, Triamcinolone hexacetonides, Vancomycins, Varicella Zosterimmunoglobulins, Varicella vaccines, other vaccines, Alemtuzumabs,Alitretinoins, Allopurinols, Altretamines, Amifostines, Anastrozoles,Arsenics, Arsenic Trioxides, Asparaginases, Bacillus Calmette-Guerin(BCG) vaccines, BCG Live, Bexarotenes, Bleomycins, Busulfans, Busulfanintravenous, Busulfan orals, Calusterones, Capecitabines, Carboplatins,Carmustines, Carmustines with Polifeprosans, Celecoxibs, Chlorambucils,Cisplatins, Cladribines, Cyclophosphamides, Cytarabines, Cytarabineliposomals, Dacarbazines, Dactinomycins, Daunorubicin liposomals,Daunorubicins, Daunomycins, Denileukin Diftitoxes, Dexrazoxanes,Docetaxels, Doxorubicins, Doxorubicin liposomals, Dromostanolonepropionates, Elliott's B Solutions, Epirubicins, Epoetin alfas,Estramustines, Etoposides, Etoposide phosphates, Etoposide VP-16s,Exemestanes, Floxuridines, Fludarabines, Fluorouracils, 5-Fluorouracils,Fulvestrants, Gemcitabines, Gemtuzumabs, Ozogamicins, Gemtuzumabozogamicins, Hydroxyureas, Idarubicins, Ifosfamides, Imatinib mesylates,Irinotecans, Letrozoles, Leucovorins, Levamisoles, Lomustines, CCNUs,Mechlorethamines, Nitrogen mustards, Megestrols, Megestrol acetates,Melphalans, L-PAMs, Mercaptopurines, 6-Mercaptopurines, Mesnas,Methotrexates, Methoxsalens, Mitomycins, Mitomycin C's, Mitotanes,Mitoxantrones, Nandrolones, Nandrolone Phenpropionates, Nofetumomabs,Oprelvekins, Oxaliplatins, Paclitaxels, Pamidronates, Pegademases,Pentostatins, Pipobromans, Plicamycins, Mithramycins, Porfimers,Porfimer sodiums, Procarbazines, Quinacrines, Rasburicases, Rituximabs,Sargramostims, Streptozocins, Talcs, Tamoxifens, Temozolomides,Teniposides, Testolactones, Thioguanines, 6-Thioguanines,Triethylenethiophosphoramides (Thiotepas), Topotecans, Toremifenes,Trastuzumabs, Tretinoins, Uracil Mustards, Valrubicins, Vinblastines,Vincristines, Vinorelbines, Zoledronates, Acivicins, Aclarubicins,Acodazoles, Acronines, Adozelesins, Aldesleukins, Retinoic Acids,Alitretinoins, 9-Cis-Retinoic Acids, Alvocidibs, Ambazones, Ambomycins,Ametantrones, Aminoglutethimides, Amsacrines, Anaxirones, Ancitabines,Anthramycins, Apaziquones, Argimesnas, Asperlins, Atrimustines,Azacitidines, Azetepas, Azotomycins, Banoxantrones, Batabulins,Batimastats, Benaxibines, Bendamustines, Benzodepas, Bicalutamides,Bietaserpines, Biricodars, Bisantrenes, Bisnafide Dimesylates,Bizelesins, Bortezomibs, Brequinars, Bropirimines, Budotitanes,Cactinomycins, Canertinibs, Caracemides, Carbetimers, Carboquones,Carmofurs, Carubicins, Carzelesins, Cedefingols, Cemadotins,Chlorambucils, Cioteronels, Cirolemycins, Clanfenurs, Clofarabines,Crisnatols, Decitabines, Dexniguldipines, Dexormaplatins, Dezaguanines,Diaziquones, Dibrospidiums, Dienogests, Dinalins, Disermolides,Dofequidars, Doxifluridines, Droloxifenes, Duazomycins, Ecomustines,Edatrexates, Edotecarins, Eflornithines, Elacridars, Elinafides,Elsamitrucins, Emitefurs, Enloplatins, Enpromates, Enzastaurins,Epipropidines, Eptaloprosts, Erbulozoles, Esorubicins, Etanidazoles,Etoglucids, Etoprines, Exisulinds, Fadrozoles, Fazarabines,Fenretinides, Fluoxymesterones, Flurocitabines, Fosquidones,Fostriecins, Fotretamines, Galarubicins, Galocitabines, Geroquinols,Gimatecans, Gimeracils, Gloxazones, Glufosfamides, Ilmofosines,Ilomastats, Imexons, Improsulfans, Indisulams, Inproquones,Interleukins, Interleukin-2s, recombinant Interleukins, Intoplicines,Iobenguanes, Iproplatins, Irsogladines, Ixabepilones, Ketotrexates,L-Alanosines, Lanreotides, Lapatinibs, Ledoxantrones, Leuprolides,Leuprorelins, Lexacalcitols, Liarozoles, Lobaplatins, Lometrexols,Lonafarnibs, Losoxantrones, Lurtotecans, Mafosfamides, Mannosulfans,Marimastats, Masoprocols, Maytansines, Mechlorethamines, Melengestrols,Melphalans, Menogarils, Mepitiostanes, Metesinds, Metomidates,Metoprines, Meturedepas, Miboplatins, Miproxifenes, Misonidazoles,Mitindomides, Mitocarcins, Mitocromins, Mitoflaxones, Mitogillins,Mitoguazones, Mitomalcins, Mitonafides, Mitoquidones, Mitospers,Mitozolomides, Mivobulins, Mizoribines, Mofarotenes, Mopidamols,Mubritinibs, Mycophenolic Acids, Nedaplatins, Neizarabines,Nemorubicins, Nitracrines, Nocodazoles, Nogalamycins, Nolatrexeds,Nortopixantrones, Ormaplatins, Ortataxels, Oteracils, Oxisurans,Oxophenarsines, Patupilones, Peldesines, Peliomycins, Pelitrexols,Pemetrexeds, Pentamustines, Peplomycins, Perfosfamides, Perifosines,Picoplatins, Pinafides, Piposulfans, Pirfenidones, Piroxantrones,Pixantrones, Plevitrexeds, Plomestanes, Porfiromycins, Prednimustines,Propamidines, Prospidiums, Pumitepas, Puromycins, Pyrazofurins,Ranimustines, Riboprines, Ritrosulfans, Rogletimides, Roquinimexs,Rufocromomycins, Sabarubicins, Safingols, Satraplatins, Sebriplatins,Semustines, Simtrazenes, Sizofirans, Sobuzoxanes, Sorafenibs,Sparfosates, Sparfosic Acids, Sparsomycins, Spirogermaniums,Spiromustines, Spiroplatins, Squalamines, Streptonigrins,Streptovarycins, Sufosfamides, Sulofenurs, Tacedinalines, Talisomycins,Tallimustines, Tariquidars, Tauromustines, Tecogalans, Tegafurs,Teloxantrones, Temoporfins, Teroxirones, Thiamiprines, Tiamiprines,Tiazofurins, Tilomisoles, Tilorones, Timcodars, Timonacics,Tirapazamines, Topixantrones, Trabectedins, Ecteinascidin 743,Trestolones, Triciribines, Trilostanes, Trimetrexates, TriplatinTetranitrates, Triptorelins, Trofosfamides, Tubulozoles, Ubenimexs,Uredepas, Valspodars, Vapreotides, Verteporfins, Vinblastines,Vindesines, Vinepidines, Vinflunines, Vinformides, Vinglycinates,Vinleucinols, Vinleurosines, Vinrosidines, Vintriptols, Vinzolidines,Vorozoles, Xanthomycin A's, Guamecyclines, Zeniplatins, Zilascorbs[2-H], Zinostatins, Zorubicins, Zosuquidars, Acetazolamides, Acyclovirs,Adipiodones, Alatrofloxacins, Alfentanils, Allergenic extracts, Alpha1-proteinase inhibitors, Alprostadils, Amikacins, Amino acids,Aminocaproic acids, Aminophyllines, Amitriptylines, Amobarbitals,Amrinones, Analgesics, Anti-poliomyelitic vaccines, Anti-rabic serums,Anti-tetanus immunoglobulins, tetanus vaccines, Antithrombin IIIs,Antivenom serums, Argatrobans, Arginines, Ascorbic acids, Atenolols,Atracuriums, Atropines, Aurothioglucoses, Azathioprines, Aztreonams,Bacitracins, Baclofens, Basiliximabs, Benzoic acids, Benztropines,Betamethasones, Biotins, Bivalirudins, Botulism antitoxins, Bretyliums,Bumetanides, Bupivacaines, Buprenorphines, Butorphanols, Calcitonins,Calcitriols, Calciums, Capreomycins, Carboprosts, Carnitines,Cefamandoles, Cefoperazones, Cefotaximes, Cefoxitins, Ceftizoximes,Cefuroximes, Chloramphenicols, Chloroprocaines, Chloroquines,Chlorothiazides, Chlorpromazines, Chondroitinsulfuric acids,Choriogonadotropin alfas, Chromiums, Cidofovirs, Cimetidines,Ciprofloxacins, Cisatracuriums, Clonidines, Codeines, Colchicines,Colistins, Collagens, Corticorelin ovine triflutates, Corticotrophins,Cosyntropins, Cyanocobalamins, Cyclosporines, Cysteines, Dacliximabs,Dalfopristins, Dalteparins, Danaparoids, Dantrolenes, Deferoxamines,Desmopressins, Dexamethasones, Dexmedetomidines, Dexpanthenols,Dextrans, Iron dextrans, Diatrizoic acids, Diazepams, Diazoxides,Dicyclomines, Digibinds, Digoxins, Dihydroergotamines, Diltiazems,Diphenhydramines, Dipyridamoles, Dobutamines, Dopamines, Doxacuriums,Doxaprams, Doxercalciferols, Doxycyclines, Droperidols, Dyphyllines,Edetic acids, Edrophoniums, Enalaprilats, Ephedrines, Epoprostenols,Ergocalciferols, Ergonovines, Ertapenems, Erythromycins, Esmolols,Estradiols, Estrogenics, Ethacrynic acids, Ethanolamines, Ethanols,Ethiodized oils, Etidronic acids, Etomidates, Famotidines, Fenoldopams,Fentanyls, Flumazenils, Fluoresceins, Fluphenazines, Folic acids,Fomepizoles, Fomivirsens, Fondaparinuxs, Foscarnets, Fosphenytoins,Furosemides, Gadoteridols, Gadoversetamides, Ganciclovirs, Gentamicins,Glucagons, Glucoses, Glycines, Glycopyrrolates, Gonadorelins,Gonadotropin chorionics, Haemophilus B polysaccharides, Hemins, Herbals,Histamines, Hydralazines, Hydrocortisones, Hydromorphones,Hydroxocobalamins, Hydroxyzines, Hyoscyamines, Ibutilides,Imiglucerases, Indigo carmines, Indomethacins, Iodides, Iopromides,lothalamic acids, loxaglic acids, Ioxilans, Isoniazids, Isoproterenols,Japanese encephalitis vaccines, Kanamycins, Ketamines, Labetalols,Lepirudins, Levobupivacaines, Levothyroxines, Lincomycins,Liothyronines, Luteinizing hormones, Lyme disease vaccines,Mangafodipirs, Manthtols, Meningococcal polysaccharide vaccines,Meperidines, Mepivacaines, Mesoridazines, Metaraminols, Methadones,Methocarbamols, Methohexitals, Methyldopates, Methylergonovines,Metoclopramides, Metoprolols, Metronidazoles, Minocyclines, Mivacuriums,Morrhuic acids, Moxifloxacins, Muromonab-CD3s, Mycophenolate mofetils,Nafcillins, Nalbuphines, Nalmefenes, Naloxones, Neostigmines,Niacinamides, Nicardipines, Nitroglycerins, Nitroprussides,Norepinephrines, Orphenadrines, Oxacillins, Oxymorphones,Oxytetracyclines, Oxytocins, Pancuroniums, Panthenols, Pantothenicacids, Papaverines, Peginterferon alpha 2As, Penicillin Gs,Pentamidines, Pentazocines, Pentobarbitals, Perflutrens, Perphenazines,Phenobarbitals, Phentolamines, Phenylephrines, Phenytoins,Physostigmines, Phytonadiones, Polymyxin, Pralidoximes, Prilocaines,Procainamides, Procaines, Prochlorperazines, Progesterones,Propranolols, Pyridostigmine hydroxides, Pyridoxines, Quinidines,Quinupristins, Rabies immunoglobulins, Rabies vaccines, Ranitidines,Remifentanils, Riboflavins, Rifampins, Ropivacaines, Samariums,Scopolamines, Seleniums, Sermorelins, Sincalides, Somatrems,Spectinomycins, Streptokinases, Streptomycins, Succinylcholines,Sufentanils, Sulfamethoxazoles, Tacrolimuses, Terbutalines,Teriparatides, Testosterones, Tetanus antitoxins, Tetracaines,Tetradecyl sulfates, Theophyllines, Thiamines, Thiethylperazines,Thiopentals, Thyroid stimulating hormones, Tinzaparins, Tirofibans,Tobramycins, Tolazolines, Tolbutamides, Torsemides, Tranexamic acids,Treprostinils, Trifluoperazines, Trimethobenzamides, Trimethoprims,Tromethamines, Tuberculins, Typhoid vaccines, Urofollitropins,Urokinases, Valproic acids, Vasopressins, Vecuroniums, Verapamils,Voriconazoles, Warfarins, Yellow fever vaccines, Zidovudines, Zincs,Ziprasidone hydrochlorides, Aclacinomycins, Actinomycins, Adriamycins,Azaserines, 6-Azauridines, Carzinophilins, Chromomycins, Denopterins, 6Diazo 5 Oxo-L-Norleucines, Enocitabines, Floxuridines, Olivomycins,Pirarubicins, Piritrexims, Pteropterins, Tegafurs, Tubercidins,Alteplases, Arcitumomabs, bevacizumabs, Botulinum Toxin Type A's,Botulinum Toxin Type B's, Capromab Pendetides, Daclizumabs, Dornasealphas, Drotrecogin alphas, Imciromab Pentetates, Iodine-131's, anantibiotic agent; an angiogenesis inhibitor; anti-cataract andanti-diabetic retinopathy substances; carbonic anhydrase inhibitors;mydriatics; photodynamic therapy agents; prostaglandin analogs; growthfactor; anti-neoplastics; anti-metabolites; anti-viral; amebicides andanti-protozoals; anti-tuberculosis and anti-leprotic; antitoxins andantivenins; antihemophilic factor, anti-inhibitor coagulant complex,antithrombin III, coagulations Factor V, coagulation Factor IX, plasmaprotein fraction, von Willebrand factor; antiplatelet agent a colonystimulating factor (CSF); an erythropoiesis stimulator; hemostatics andalbumins; Immune Globulins; thrombin inhibitors; anticoagulants; asteroidal anti-inflammatory drug selected from among alclometasones,algestones, beclomethasones, betamethasones, budesonides, clobetasols,clobetasones, clocortolones, cloprednols, corticosterones, cortisones,cortivazols, deflazacorts, desonides, desoximetasones, dexamethasones,diflorasones, diflucortolones, difluprednates, enoxolones, fluazacorts,flucloronides, flumethasones, flunisolides, fluocinolones,fluocinonides, fluocortins, fluocortolones, fluorometholones,fluperolones, fluprednidenes, fluprednisolones, flurandrenolides,fluticasones, formocortals, halcinonides, halobetasols, halometasones,halopredones, hydrocortamates, hydrocortisones, loteprednol etabonate,mazipredones, medrysones, meprednisones, methylprednisolones, mometasonefuroate, paramethasones, prednicarbates, prednisolones, prednisones,prednivals, prednylidenes, rimexolones, tixocortols and triamcinolones;Docosanols, prostaglandins, prostaglandin analogs, antiprostaglandinsand prostaglandin precursors; miotics, cholinergics andanti-cholinesterase; and anti-allergenics.

The compositions and modified PH20 polypeptides can be used to treat anycondition normally treated by the PH20 polypeptide or thetherapeutically active agent. These include, for example, conditions inwhich hyaluronan plays a role or is associated with the etiology of thedisease due to, for example, accumulation or overproduction ofhyaluronan. Hence provided are methods, uses of the compositions andmodified PH20 polypeptides for treating a hyaluronan-associated diseaseor condition by administering any of the modified PH20 polypeptides orcompositions provided herein. Hyaluronan-associated diseases andconditions include, for example, inflammatory disease and tumors orcancers, including a late-stage cancer, metastatic cancers andundifferentiated cancers, such as ovarian cancer, in situ carcinoma(ISC), squamous cell carcinoma (SCC), prostate cancer, pancreaticcancer, non-small cell lung cancer, breast cancer and colon cancer. ThePH20 polypeptide can be modified to exhibit increased half-life for suchtreatments. For example, the PH20 polypeptide can be modified with apolymer such as a PEG moiety for such treatments.

Also provided are methods for increasing delivery of a therapeutic agentto a subject by: administering to a subject any of the modified PH20polypeptides or compositions provided herein, and administering thetherapeutic agent. The therapeutic agent can be administered in the samecomposition or separately, and can be administered before or after,simultaneously, or intermittently, with administration of the PH20polypeptide(s). Administration includes any route, including intravenousand subcutaneous administration, such as simultaneously with,intermittently with, or subsequent to administration of the therapeuticagent. The therapeutic agents include any of those set forth above,elsewhere herein and/or known to those of skill in the art.

Also provided are methods for treating an excess of glycosaminoglycans;for treating a tumor; for treating glycosaminoglycan accumulation in thebrain; for treating a cardiovascular disorder; for treating anophthalmic disorder; for treating pulmonary disease; for increasingpenetration of chemotherapeutic agents into solid tumors; for treatingcellulite; for treating a proliferative disorder; or for increasingbioavailability of drugs and other therapeutic agents by administeringthe modified PH20 polypeptides or compositions provided herein.

Also provided are pharmaceutical compositions for use in treating ahyaluronan-associated disease or disorder; for use in delivering atherapeutic agent to a subject; for treating an excess ofglycosaminoglycans; for treating a tumor; for treating glycosaminoglycanaccumulation in the brain; for treating a cardiovascular disorder; fortreating an ophthalmic disorder; for treating pulmonary disease; forincreasing penetration of chemotherapeutic agents into solid tumors; fortreating cellulite; for treating a proliferative disorder; or forincreasing bioavailability of drugs and other therapeutic agents; andfor any other use of compositions containing PH20 polypeptides.

Provided herein is a method for identifying or selecting a modifiedhyaluronan-degrading enzyme that exhibits stability under a denaturationcondition that includes the steps of: a) testing the activity of amodified hyaluronan-degrading enzyme in a composition containing adenaturing agent and/or under a denaturing condition; b) testing theactivity of the modified hyaluronan-degrading enzyme in the samecomposition and/or under the same conditions as a) except absent thedenaturing agent or condition; and c) selecting or identifying amodified hyaluronan-degrading enzyme that exhibits activity in a) thatis at least 5% of the activity in b). In such an example, the activityis hyaluronidase activity. In some examples of the methods, a modifiedhyaluronan-degrading enzyme is selected or identified if the activity ina) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the activity in b), forexample, a modified hyaluronan-degrading enzyme is selected oridentified if the activity in a) is at least 40% or more of the activityin b). The method also can include steps of: d) comparing the activityof the modified hyaluronan-degrading enzyme in a) to the activity of theunmodified hyaluronan-degrading enzyme tested under the same conditions;and e) identifying or selecting a modified hyaluronan-degrading enzymethat exhibits at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%,180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%,5000% or more of the hyaluronidase activity compared to the unmodifiedhyaluronan-degrading enzyme.

Also provided herein is a method for identifying or selecting a modifiedhyaluronan-degrading enzyme that exhibits stability, such as increasedstability, under a denaturation condition, that includes the steps of:a) testing the activity of a modified hyaluronan-degrading enzyme in acomposition containing a denaturing agent and/or under a denaturingcondition; b) testing the activity of the corresponding unmodifiedhyaluronan-degrading enzyme in a composition containing the samedenaturing agent and/or under the same denaturing condition as a),whereby the activity is tested under the same conditions as a); and c)selecting or identifying a modified hyaluronan-degrading enzyme thatexhibits greater activity than the unmodified hyaluronan-degradingenzyme, thereby identifying or selecting a modified hyaluronan-degradingenzyme that exhibits increased stability under a denaturation condition.In such an example, the activity can be a hyaluronidase activity. Inexamples of the method, a modified hyaluronan-degrading enzyme isselected or identified if the activity is at least 120%, 130%, 135%,140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%,1500%, 2000%, 3000%, 4000%, 5000% or more of the activity compared tothe unmodified hyaluronan-degrading enzyme. In such an example, themethod also can include additional steps of: d) testing the activity ofthe selected or identified modified hyaluronan-degrading enzyme in acomposition containing a denaturing agent and/or under a denaturingcondition; e) testing the activity of the same selected or identifiedmodified hyaluronan-degrading enzyme in the same composition and/orunder the same conditions as d) except absent the denaturing agent orcondition; and f) selecting or identifying a modifiedhyaluronan-degrading enzyme that exhibits activity in d) that is atleast 5% of the activity in e). In such an example, the activity ishyaluronidase activity. In some examples of the methods, a modifiedhyaluronan-degrading enzyme is selected or identified if the activity ind) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the activity in e), forexample, a modified hyaluronan-degrading enzyme is selected oridentified if the activity in d) is at least 40% or more of the activityin e).

In any of the methods provided herein for identifying or selecting amodified hyaluronan-degrading enzyme, the denaturing agent or conditionis caused by temperature, agitation, no or low salt or the presence ofan excipient. For example, the denaturing agent or condition is causedby elevated temperature that is from or from about 30° C. to 42° C.,such as greater than or greater than about 30° C., 31° C., 32° C., 33°C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C. or42° C. In other examples, the denaturing agent or condition is theabsence of salt or low salt less than 100 mM, such as low salt less than90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10mM, 5 mM. In further examples, the denaturing agent or condition is adenaturing excipient selected from among an antiadherents, binders,coatings, fillers and diluents, flavors, colors, lubricants, glidants,preservatives, sorbents and sweeteners.

In particular examples of any of the methods provided herein foridentifying or selecting a modified hyaluronan-degrading enzyme, thedenaturing agent or condition is a preservative(s), for example, aphenolic preservative(s). The phenolic preservative(s) can be a phenol,metacresol (m-cresol), benzyl alcohol, or a paraben. For example, thedenaturing agent or condition is a preservative(s) that is phenol and/orm-cresol. In such examples, the total amount of phenolic preservative inthe composition, as a percentage (%) of mass concentration (w/v), isfrom or from about 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4%inclusive.

In any of the methods provided herein for identifying or selecting amodified hyaluronan-degrading enzyme, prior to testing the activity of ahyaluronan-degrading enzyme composition in a) and/or b), thehyaluronan-degrading enzyme is exposed to the denaturation condition ordenaturing agent for a predetermined time. The predetermined time is atime period that is user selected depending on the particularhyaluronan-degrading enzyme that is being evolved or selected, theparticular denaturation condition or denaturing agent, the amount orextent of the denaturation condition or denaturing agent, theapplication or use of the hyaluronan-degrading enzyme and other similarfactors. For example, the predetermined time can be from or from about 1minute to 1 month, 1 minute to 3 weeks, 1 minute to 2 weeks, 1 minute to1 week, 1 minute to 24 hours, 1 minute to 12 hours, 30 minutes to 6hours or 1 hour to 4 hours, such as at least or about at least 30minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, two days, threedays, four days, five days, six days, 7 days, two weeks or one month.

In any of the methods provided herein for identifying or selecting amodified hyaluronan-degrading enzyme, the modified hyaluronan-degradingenzyme is one that contains an amino acid replacement, insertion ordeletion of amino acids compared to an unmodified hyaluronan-degradingenzyme. For example, the modified hyaluronan-degrading enzyme containsan amino acid replacement, such as a single amino acid replacement ortwo, three, four, five, six, seven, eight, nine or more amino acidreplacements compared to an unmodified form of the hyaluronan-degradingenzyme. In particular aspects of the method, a library or collection ofmodified hyaluronan-degrading enzymes are screened in order to evolve oridentify or select a modified hyaluronan-degrading enzyme that exhibitsstability, such as increased stability, under a denaturation condition.Thus, in examples of the methods herein, a plurality of modifiedhyaluronan-degrading enzymes are tested in a) and/or b). In suchexamples, the plurality of modified hyaluronan-degrading enzymes aremodified compared to the corresponding unmodified hyaluronan-degradingenzyme to generate a collection of modified hyaluronan-degradingenzymes, whereby each modified protein in the collection is tested ineach of a) and/or b). In the collection or library, each modifiedhyaluronan-degrading enzyme contains a single amino acid replacementcompared to the unmodified form of the hyaluronan-degrading enzyme, suchthat the plurality of modified enzymes are such that the amino acid ateach modified position is replaced by up to 1-19 other amino acids otherthan the original amino acid at the position, whereby each modifiedhyaluronan-degrading enzyme contains a different amino acid replacement,and every amino acid along the length of the hyaluronan-degradingenzyme, or a selected portion thereof, is replaced.

In any of the methods provided herein, the modified hyaluronan-degradingenzyme is modified compared to an unmodified hyaluronan-degrading enzymeby insertion, deletion or replacement of an amino acid(s). Theunmodified hyaluronan-degrading enzyme can be a chondroitinase or can bea hyaluronidase. In examples herein, the unmodified hyaluronidase is aPH20 hyaluronidase or truncated form thereof lacking a C-terminalglycosylphosphatidylinositol (GPI) anchor attachment site or a portionof the GPI anchor attachment site, whereby the truncated form exhibitshyaluronidase activity. PH20 hyaluronidase can be a human, monkey,bovine, ovine, rat, fox, mouse or guinea pig PH20. In particularexamples, the PH20 hyaluronidase is a human PH20 or a C-terminaltruncated form thereof. For example, the unmodified hyaluronan-degradingenzyme is one that has the sequence of amino acids set forth in any ofSEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, 870 or asequence of amino acids that is at least 80% sequence identity to any ofSEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, 870,such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 3, 7, 10, 12,14, 24, 32-66, 69, 72, 857, 859, 861, or 870. In particular examples,the unmodified hyaluronan-degrading enzyme is a PH20 hyaluronidasehaving the sequence of amino acids set forth in any of SEQ ID NOs: 3, 7,32-66, 69 or 72, or a sequence of amino acids that exhibits at least 85%sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72, such as asequence of amino acids that exhibits at least 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toany of SEQ ID NOs: 3, 7, 32-66, 69 or 72.

In any of the methods provided herein for identifying or selecting amodified hyaluronan-degrading enzyme that exhibits stability, the methodis performed in vitro. Also provided are any of the methods that areiterative, whereby the steps of the method are repeated a plurality oftimes, wherein in each repetition, further modified hyaluronan-degradingenzymes of a selected modified hyaluronan-degrading enzyme are generatedand tested, whereby the modified hyaluronan-degrading enzyme is evolvedto exhibit increased stability under a denaturation condition. Alsoprovided herein is a modified hyaluronan-degrading enzyme identified byany of the methods provided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the amino acid sequence of full-length human PH20 (setforth in SEQ ID NO:7) and soluble C-terminal truncated variants thereof.The C-terminal amino acid residue of exemplary C-terminal truncatedvariants of full-length PH20 are indicated by bold font. The completeamino acid sequences of exemplary C-terminal truncated variants offull-length PH20 also are provided in SEQ ID NOs: 3 and 32-66. TheC-terminal amino acid residue of an exemplary soluble PH20, whosecomplete sequence is set forth in SEQ ID NO:3, also is indicated byunderline. Exemplary, non-limiting, positions for amino acidreplacements are indicated by highlighting. Corresponding positions canbe identified by alignment of a sequence of interest with any of SEQ IDNOs: 3, 7 or 32-66, and in particular with SEQ ID NO:3.

FIG. 2 (A-L) depicts exemplary alignments of human soluble PH20 setforth in SEQ ID NO:3 with other PH20 polypeptides. A “*” means that thealigned residues are identical, a “:” means that aligned residues arenot identical, but are similar and contain conservative amino acidsresidues at the aligned position, and a “.” means that the alignedresidues are similar and contain semi-conservative amino acid residuesat the aligned position. Exemplary, non-limiting, correspondingpositions for amino acid replacements are indicated by highlighting. Forexample, FIG. 2A depicts the alignment of a human soluble PH20 set forthin SEQ ID NO:3 with chimpanzee PH20 set forth in SEQ ID NO:10. FIG. 2Bdepicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3with Rhesus monkey PH20 set forth in SEQ ID NO:12. FIG. 2C depicts thealignment of a human soluble PH20 set forth in SEQ ID NO:3 withCynomolgus monkey PH20 set forth in SEQ ID NO:14. FIG. 2D depicts thealignment of human soluble PH20 set forth in SEQ ID NO:3 with bovinePH20 set forth in SEQ ID NO:16. FIG. 2E depicts the alignment of a humansoluble PH20 set forth in SEQ ID NO:3 with mouse PH20 set forth in SEQID NO:20. FIG. 2F depicts the alignment of a human soluble PH20 setforth in SEQ ID NO:3 with rat PH20 set forth in SEQ ID NO:22. FIG. 2Gdepicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3with rabbit PH20 set forth in SEQ ID NO:24. FIG. 2H depicts thealignment of a human soluble PH20 set forth in SEQ ID NO:3 with guineapig PH20 set forth in SEQ ID NO:29. FIG. 2I depicts the alignment of ahuman soluble PH20 set forth in SEQ ID NO:3 with Fox PH20 set forth inSEQ ID NO:31. FIG. 2J depicts the alignment of a human soluble PH20 setforth in SEQ ID NO:3 with Gibbon PH20 set forth in SEQ ID NO:857.

FIG. 2K depicts the alignment of a human soluble PH20 set forth in SEQID NO:3 with Marmoset PH20 set forth in SEQ ID NO:859. FIG. 2L depictsthe alignment of a human soluble PH20 set forth in SEQ ID NO:3 withOrangutan PH20 set forth in SEQ ID NO:861.

DETAILED DESCRIPTION Outline

-   -   A. DEFINITIONS    -   B. PH20 HYALURONIDASE        -   1. Structure        -   2. Function        -   3. Soluble PH20 Polypeptides    -   C. MODIFIED PH20 POLYPEPTIDES        -   1. Active Mutants            -   a. Increased Activity            -   b. Increased Stability                -   i. Phenophiles                -   ii. Thermophiles                -   iii. Absence of Salt        -   2. Inactive Mutants        -   3. Additional Modifications and Conjugates            -   a. Decreased Immunogenicity            -   b. Conjugation to Polymers    -   D. METHODS FOR IDENTIFYING MODIFIED HYALURONAN-DEGRADING ENZYMES        WITH ALTERED PROPERTIES OR ACTIVITIES        -   1. Hyaluronan-Degrading Enzymes and Libraries of Modified            Hyaluronan-Degrading Enzymes        -   2. Screening or Testing for a Desired Activity or Property        -   3. Selection or Identification        -   4. Iterative Methods    -   E. PRODUCTION OF MODIFIED POLYPEPTIDES AND ENCODING NUCLEIC ACID        MOLECULES        -   1. Isolation or Preparation of Nucleic Acids Encoding PH20            Polypeptides        -   2. Generation of Mutant or Modified Nucleic Acid and            Encoding Polypeptides        -   3. Vectors and Cells        -   4. Expression            -   a. Prokaryotic Cells            -   b. Yeast Cells            -   c. Insects and Insect Cells            -   d. Mammalian expression            -   e. Plants and plant cells        -   5. Purification        -   6. Modification of Polypeptides by PEGylation    -   F. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS, DOSAGES AND        ADMINISTRATION        -   1. Formulations—liquids, injectables, solutions and            emulsions            -   a. Lyophilized Powders            -   b. Exemplary Formulations                -   i. Salt (e.g. NaCl)                -   ii. pH and Buffer                -   iii. Preservative(s)                -   iv. Stabilizers        -   2. Compositions for Other Routes of Administration        -   3. Dosages and Administration        -   4. Exemplary PH20-Insulin Co-Formulations        -   5. Packaging, Articles of Manufacture and Kits    -   G. METHODS OF ASSESSING PH20 ACTIVITY AND STABILITY        -   1. Hyaluronidase Activity        -   2. Solubility        -   3. Purity, Crystallization or Aggregation        -   4. Pharmacodynamics/Pharmacokinetics    -   H. METHODS OF TREATMENT AND COMBINATION THERAPY        -   1. Methods of Delivering Therapeutic Agents            -   Delivery of Insulin        -   2. Methods of Treating Hyaluronan-Associated Disease and            Conditions (eg., Tumors)        -   3. Other Uses        -   4. Contraception    -   I. EXAMPLES

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, GenBank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, a hyaluronan-degrading enzyme refers to an enzyme thatcatalyzes the cleavage of a hyaluronan polymer (also referred to ashyaluronic acid or HA) into smaller molecular weight fragments.Exemplary hyaluronan-degrading enzymes are hyaluronidases, andparticular chondroitinases and lyases that have the ability todepolymerize hyaluronan. Exemplary chondroitinases that arehyaluronan-degrading enzymes include, but are not limited to,chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AClyase (also known as chondroitin sulfate lyase or chondroitin sulfateeliminase) and chondroitin C lyase. Chondroitin ABC lyase contains twoenzymes, chondroitin-sulfate-ABC endolyase (EC 4.2.2.20) andchondroitin-sulfate-ABC exolyase (EC 4.2.2.21). An exemplarychondroitin-sulfate-ABC endolyases and chondroitin-sulfate-ABC exolyasesinclude, but are not limited to, those from Proteus vulgaris andPedobacter heparinus (the Proteus vulgaris chondroitin-sulfate-ABCendolyase is set forth in SEQ ID NO:922; Sato et al. (1994) Appl.Microbiol. Biotechnol. 41(1):39-46). Exemplary chondroitinase AC enzymesfrom bacteria include, but are not limited to, those from Pedobacterheparinus, set forth in SEQ ID NO: 923, Victivallis vadensis, set forthin SEQ ID NO:924, and Arthrobacter aurescens (Tkalec et al. (2000)Applied and Environmental Microbiology 66(1):29-35; Ernst et al. (1995)Critical Reviews in Biochemistry and Molecular Biology 30(5):387-444).Exemplary chondroitinase C enzymes from bacteria include, but are notlimited to, those from Streptococcus and Flavobacterium (Hibi et al.(1989) FEMS-Microbiol-Lett. 48(2): 121-4; Michelacci et al. (1976) J.Biol. Chem. 251:1154-8; Tsuda et al. (1999) Eur. 1 Biochem.262:127-133).

As used herein, hyaluronidase refers to a class of enzymes that degradehyaluronan. Hyaluronidases include, but are not limited to, bacterialhyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidases from leeches,other parasites and crustaceans (EC 3.2.1.36), and mammalian-typehyaluronidases (EC 3.2.1.35). Hyaluronidases include any of non-humanorigin including, but not limited to, murine, canine, feline, leporine,avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, andany from leeches, other parasites, and crustaceans. Exemplary humanhyaluronidases include HYAL1, HYAL2, HYAL3, HYAL4, and PH20. Alsoincluded amongst hyaluronidases are soluble hyaluronidases, including,ovine and bovine PH20, and soluble PH20. Exemplary hyaluronidasesinclude any set forth in SEQ ID NOs: 6, 7-31, 69, 70, 71, 72, 856-861,869-921, mature forms thereof (lacking the signal sequence), or allelicor species variants thereof. Hyaluronidases also include truncated formsthereof that exhibit hyaluronidase activity, including C-terminaltruncated variants that are soluble.

As used herein, PH20 refers to a type of hyaluronidase that occurs insperm and is neutral-active. PH-20 occurs on the sperm surface, and inthe lysosome-derived acrosome, where it is bound to the inner acrosomalmembrane. PH20 includes those of any origin including, but not limitedto, human, chimpanzee, Cynomolgus monkey, Rhesus monkey, murine, bovine,ovine, guinea pig, rabbit and rat origin. Exemplary PH20 polypeptides,including precursor and mature forms, include those from human (SEQ IDNOs:6 and 7), chimpanzee (SEQ ID NOs:8, 9, 10, 869 and 870), Rhesusmonkey (SEQ ID NOs:11 and 12), Cynomolgus monkey (SEQ ID NOs:13 and 14),cow (e.g., SEQ ID NOs:15-18); mouse (SEQ ID NOs:19 and 20); rat (SEQ IDNOs:21 and 22); rabbit (SEQ ID NOs:23 and 24); sheep (SEQ ID NOs:25-27),guinea pig (SEQ ID NOs:28 and 29); fox (SEQ ID NOs: 30 and 31); Gibbon(SEQ ID NOs:856 and 857), Marmoset (SEQ ID NOs:858 and 859) andorangutan (SEQ ID NOs:860 and 861). Reference to PH20 includes precursorPH20 polypeptides and mature PH20 polypeptides (such as those in which asignal sequence has been removed), truncated forms thereof that haveactivity, and includes allelic variants and species variants, variantsencoded by splice variants, and other variants, including polypeptidesthat have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more sequence identity to the precursorpolypeptides set forth in SEQ ID NO:7, or the mature forms thereof. PH20polypeptides also include those that contain chemical orposttranslational modifications and those that do not contain chemicalor posttranslational modifications. Such modifications include, but arenot limited to, PEGylation, albumination, glycosylation, farnysylation,carboxylation, hydroxylation, phosphorylation, and other polypeptidemodifications known in the art. Examples of commercially availablebovine or ovine soluble hyaluronidases are Vitrase® hyaluronidase (ovinehyaluronidase) and Amphadase® hyaluronidase (bovine hyaluronidase).

As used herein, a soluble PH20 refers to a polypeptide characterized byits solubility under physiological conditions. Generally, a soluble PH20lacks all or a portion of a glycophosphatidyl anchor (GPI) attachmentsequence, or does not otherwise sufficiently anchor to the cellmembrane. For example, a soluble PH20 can be a C-terminally truncatedvariant of a PH20 lacking a contiguous sequence of amino acids thatcorresponds to all or a portion of a glycophosphatidyl anchor (GPI)attachment sequence. Hence, upon expression from a cell, a soluble PH20is secreted into the medium. Soluble PH20 proteins can be distinguished,for example, by its partitioning into the aqueous phase of a Triton®X-114 detergent solution warmed to 37° C. (Bordier et al., (1981) J.Biol. Chem., 256:1604-7). Membrane-anchored, such as lipid anchoredhyaluronidases, will partition into the detergent rich phase, but willpartition into the detergent-poor or aqueous phase following treatmentwith Phospholipase-C. Included among soluble PH20 hyaluronidases aremembrane anchored hyaluronidases in which one or more regions associatedwith anchoring of the hyaluronidase to the membrane has been removed ormodified, where the soluble form retains hyaluronidase activity. Solublehyaluronidases include recombinant soluble hyaluronidases and thosecontained in or purified from natural sources, such as, for example,testes extracts from sheep or cows. Exemplary of such solublehyaluronidases are soluble human PH20 (SEQ ID NOs: 3 or 32-66). Othersoluble hyaluronidases include ovine (SEQ ID NOs:25-27) and bovine (SEQID NO:16 or 18) PH20.

As used herein, soluble human PH20 (sHuPH20) includes human PH20polypeptides that lack a contiguous sequence of amino acids from theC-terminus of human PH20 that includes all or a portion of theglycosylphosphatidylinositol (GPI) anchor sequence (C-terminallytruncated PH20 polypeptides) such that upon expression, the polypeptidesare soluble under physiological conditions. For example, soluble humanPH20 polypeptides are C-terminally truncated polypeptides of human PH20set forth as SEQ ID NO:6 in its precursor form or in SEQ ID NO:7 in itsmature form lacking the signal sequence, or allelic variants thereof(e.g. set forth in any of SEQ ID NOs: 68-72). Solubility can be assessedby any suitable method that demonstrates solubility under physiologicconditions. Exemplary of such methods is the Triton® X-114 assay, thatassesses partitioning into the aqueous phase and that is describedabove. In addition, a soluble human PH20 polypeptide is, if produced inCHO cells, such as CHO-S cells, a polypeptide that is expressed and issecreted into the cell culture medium. Soluble human PH20 polypeptides,however, are not limited to those produced in CHO cells, but can beproduced in any cell or by any method, including recombinant expressionand polypeptide synthesis. Reference to secretion in CHO cells isdefinitional. Hence, if a polypeptide could be expressed and secreted inCHO cells and is soluble in the media, i.e., partitions into the aqueousphase when extracted with Triton® X-114 detergent, it is a soluble PH20polypeptide whether or not it is so-produced. The precursor polypeptidesfor sHuPH20 polypeptides can include a signal sequence, such as aheterologous or non-heterologous (i.e., native) signal sequence.Exemplary of the precursors are those that include a signal sequence,such as the native 35 amino acid signal sequence at amino acid positions1-35 (see, e.g., amino acids 1-35 of SEQ ID NO:6).

As used herein, “native” or “wildtype” with reference to a PH20polypeptide refers to a PH20 polypeptide encoded by a native ornaturally occurring PH20 gene, including allelic variants, that ispresent in an organism, including a human and other animals, in nature.Reference to wild-type PH20 without reference to a species is intendedto encompass any species of a wild-type PH20. Included among wild-typePH20 polypeptides are the encoded precursor polypeptide, fragmentsthereof, and processed forms thereof, such as a mature form lacking thesignal peptide as well as any pre- or post-translationally processed ormodified forms thereof. Also included among native PH20 polypeptides arethose that are post-translationally modified, including, but not limitedto, those that are modified by glycosylation, carboxylation and/orhydroxylation. The amino acid sequences of exemplary wild-type humanPH20 are set forth in SEQ ID NOs: 6 and 7 and those of allelic variants,including mature forms thereof, are set forth in SEQ ID NOs:68-72. Otheranimals produce native PH20, including, but not limited to, native orwildtype sequences set forth in any of SEQ ID NOs: 8-31, 856-861, 869 or870.

As used herein, modification is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids and nucleotides, respectively. Modificationsalso can include post-translational modifications or other changes tothe molecule that can occur due to conjugation or linkage, directly orindirectly, to another moiety. Methods of modifying a polypeptide areroutine to those of skill in the art, such as by using recombinant DNAmethodologies.

As used herein, a “modified hyaluronan-degrading enzyme” refers to ahyaluronan-degrading enzyme that contains a modification compared to areference or unmodified hyaluronan-degrading enzyme. The modificationcan be an amino acid replacement (substitution), insertion (addition) ordeletion of one or more amino acid residues. The amino acid residue canbe a natural or non-natural amino acid. In some cases, the modificationcan be a post-translational modification. A modifiedhyaluronan-degrading enzyme can have up to 150 amino acid differencescompared to a reference or unmodified hyaluronan-degrading enzyme, solong as the resulting modified hyaluronan-degrading enzyme exhibitshyaluronidase activity. Typically, a modified hyaluronan-degradingenzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aminoacid modifications.

As used herein, an unmodified hyaluronan-degrading enzyme refers to astarting polypeptide that is selected for modification as providedherein. The starting polypeptide can be a naturally-occurring, wild-typeform of a polypeptide. In addition, the starting polypeptide can bealtered or mutated, such that it differs from a native wild type isoformbut is nonetheless referred to herein as a starting unmodifiedpolypeptide relative to the subsequently modified polypeptides producedherein. Thus, existing proteins known in the art that have been modifiedto have a desired increase or decrease in a particular activity orproperty compared to an unmodified reference protein can be selected andused as the starting unmodified polypeptide. For example, a protein thathas been modified from its native form by one or more single amino acidchanges and possesses either an increase or decrease in a desiredproperty, such as a change in an amino acid residue or residues to alterglycosylation, can be selected for modification, and hence referred toherein as unmodified, for further modification. An unmodifiedhyaluronan-degrading enzyme includes human and non-humanhyaluronan-degrading enzymes, including hyaluronan-degrading enzymesfrom non-human mammals and bacteria. Exemplary unmodifiedhyaluronan-degrading enzyme are any set forth in SEQ ID NOs: 2, 3, 6,7-66, 68-72, 856-861, 869-924 or mature, C-terminally truncated formsthereof that exhibit hyaluronidase activity, or a hyaluronan-degradingenzyme that exhibits at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to anyof SEQ ID NOs: 2, 3, 6, 7-66, 68-72, 856-861, 869-924. It is understoodthat an unmodified hyaluronan-degrading enzyme generally is one thatdoes not contain the modification(s), such as amino acid replacement(s)of a modified hyaluronan-degrading enzyme.

As used herein, “modified PH20 polypeptide” or “variant PH20polypeptide” refers to a PH20 polypeptide that contains at least oneamino acid modification, such as at least one amino acid replacement asdescribed herein, in its sequence of amino acids compared to a referenceunmodified PH20 polypeptide. A modified PH20 polypeptide can have up to150 amino acid replacements, so long as the resulting modified PH20polypeptide exhibits hyaluronidase activity. Typically, a modified PH20polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50amino acid replacements. It is understood that a modified PH20polypeptide also can include any one or more other modifications, inaddition to at least one amino acid replacement as described herein.

As used herein, an unmodified PH20 polypeptide refers to a starting PH20polypeptide that is selected for modification as provided herein. Thestarting polypeptide can be a naturally-occurring, wild-type form of apolypeptide. In addition, the starting polypeptide can be altered ormutated, such that it differs from a native wild type isoform but isnonetheless referred to herein as a starting unmodified polypeptiderelative to the subsequently modified polypeptides produced herein.Thus, existing proteins known in the art that have been modified to havea desired increase or decrease in a particular activity or propertycompared to an unmodified reference protein can be selected and used asthe starting unmodified polypeptide. For example, a protein that hasbeen modified from its native form by one or more single amino acidchanges and possesses either an increase or decrease in a desiredproperty, such as a change in an amino acid residue or residues to alterglycosylation, can be selected for modification, and hence referred toherein as unmodified, for further modification. Exemplary unmodifiedPH20 polypeptides is a human PH20 polypeptide or allelic or speciesvariants thereof or other variants, including mature and precursorpolypeptides. For example, exemplary reference PH20 polypeptides is amature full length PH20 polypeptide set forth in SEQ ID NOs: 7, 69 or72, or in C-terminally truncated forms thereof such as set forth in anyof SEQ ID NOs: 3 and 32-66, or in a PH20 polypeptide that exhibits atleast 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ IDNOs: 3, 7, 32-66, 69 or 72. A reference PH20 polypeptide also caninclude the corresponding precursor form such as set forth in any of SEQID NOs: 2, 6, 68, 70, 71 or other precursor forms, or in a PH20polypeptide that exhibits at least 68%, 69%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to any of SEQ ID NOs: 2, 6, 68, 70, 71. It isunderstood that an unmodified hyaluronan-degrading enzyme generally isone that does not contain the modification(s), such as amino acidreplacement(s) of a modified hyaluronan-degrading enzyme.

As used herein, an N-linked moiety refers to an asparagine (N) aminoacid residue of a polypeptide that is capable of being glycosylated bypost-translational modification of a polypeptide. Exemplary N-linkedmoieties of human PH20 include amino acids N47, N131, N200, N219, N333,N358 and N365 of the sequence of amino acids set forth in SEQ ID NO: 3or 7 (corresponding to amino acid residues N82, N166, N235, N254, N368,N393 and N490 of human PH20 set forth in SEQ ID NO: 6).

As used herein, an N-glycosylated polypeptide refers to a PH20polypeptide containing oligosaccharide linkage of at least threeN-linked amino acid residues, for example, N-linked moietiescorresponding to amino acid residues N200, N333 and N358 of SEQ ID NO:3or 7. An N-glycosylated polypeptide can include a polypeptide wherethree, four, five and up to all of the N-linked moieties are linked toan oligosaccharide. The N-linked oligosaccharides can includeoligomannose, complex, hybrid or sulfated oligosaccharides, or otheroligosaccharides and monosaccharides.

As used herein, an N-partially glycosylated polypeptide refers to apolypeptide that minimally contains an N-acetylglucosamine glycan linkedto at least three N-linked moieties. A partially glycosylatedpolypeptide can include various glycan forms, including monosaccharides,oligosaccharides, and branched sugar forms, including those formed bytreatment of a polypeptide with EndoH, EndoF1, EndoF2 and/or EndoF3.

As used herein, “conditions” refers to any parameter that can influencethe activity or properties of a protein or agent. For purposes herein,conditions generally refer to the presence, including amount, ofexcipients, carriers or other components in a formulation other than theactive agent (e.g., modified PH20 hyaluronidase); temperature; time(e.g., time of storage or exposure); storage vessel; properties ofstorage (e.g., agitation) and/or other conditions associated withexposure or use.

As used herein, “denaturation” or “denaturing” or grammatical variationsthereof with reference to a protein refers to a biochemical change in aprotein so that a property or activity of the protein is diminished oreliminated. The biochemical change can be a change in the tertiarystructure of the protein to unfold. The property or activity can becompletely abolished or can be reduced by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or more.

As used herein, property refers to a physical or structural property,such as the three-dimensional structure, pI, half-life, conformation andother such physical characteristics. For example, a change in a propertycan be manifested as the solubility, aggregation or crystallization of aprotein.

As used herein, activity refers to a functional activity or activitiesof a polypeptide or portion thereof associated with a full-length(complete) protein. Functional activities include, but are not limitedto, biological activity, catalytic or enzymatic activity, antigenicity(ability to bind or compete with a polypeptide for binding to ananti-polypeptide antibody), immunogenicity, ability to form multimers,and the ability to specifically bind to a receptor or ligand for thepolypeptide.

As used herein, hyaluronidase activity refers to the ability toenzymatically catalyze the cleavage of hyaluronic acid. The UnitedStates Pharmacopeia (USP) XXII assay for hyaluronidase determineshyaluronidase activity indirectly by measuring the amount of highermolecular weight hyaluronic acid, or hyaluronan, (HA) substrateremaining after the enzyme is allowed to react with the HA for 30 min at37° C. (USP XXII-NF XVII (1990) 644-645 United States PharmacopeiaConvention, Inc, Rockville, Md.). A Reference Standard solution can beused in an assay to ascertain the relative activity, in units, of anyhyaluronidase. In vitro assays to determine the hyaluronidase activityof hyaluronidases, such as PH20, including modified PH20 polypeptides,are known in the art and described herein. Exemplary assays include themicroturbidity assay described herein that measures cleavage ofhyaluronic acid by hyaluronidase indirectly by detecting the insolubleprecipitate formed when the uncleaved hyaluronic acid binds with serumalbumin. Reference Standards can be used, for example, to generate astandard curve to determine the activity in Units of the hyaluronidasebeing tested.

As used herein, neutral active refers to the ability of a PH20polypeptide to enzymatically catalyze the cleavage of hyaluronic acid atneutral pH, such as at a pH between or about between pH 6.0 to pH 7.8.

As used herein, “increased activity” with reference to a modified PH20hyaluronidase means that, when tested under the same conditions, themodified PH20 hyaluronidase exhibits greater hyaluronidase activitycompared to an unmodified PH20 hyaluronidase not containing the aminoacid replacement(s). For example, a modified PH20 hyaluronidase exhibitsat least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%or more of the activity of the unmodified or reference PH20hyaluronidase.

As used herein, “solubility” with reference to a protein refers to aprotein that is homogenous in an aqueous solution, whereby proteinmolecules diffuse and do not sediment spontaneously. Hence a solubleprotein solution is one in which there is an absence of a visible ordiscrete particle in a solution containing the protein, such that theparticles cannot be easily filtered. Generally, a protein is soluble ifthere are no visible or discrete particles in the solution. For example,a protein is soluble if it contains no or few particles that can beremoved by a filter with a pore size of 0.22 μm.

As used herein, aggregation or crystallization with reference to aprotein refers to the presence of visible or discrete particles in asolution containing the protein. Typically, the particles are greaterthan 10 μm in size, such as greater than 15 μm, 20 μm, 25 μm, 30 μm, 40μm, 50 μm or greater. Aggregation or crystallization can arise due toreduced solubility, increased denaturation of a protein or the formationof covalent bonds.

As used herein, “denaturing condition” or “denaturation condition”refers to any condition or agent that, when exposed to a protein,affects or influences the degradation or denaturation of the protein,generally as a result of a loss or partial loss of the tertiary orsecondary structure of the protein. Denaturing conditions can result ineffects such as loss or reduction in activity, loss or reduction ofsolubility, aggregation and/or crystallization. The denaturing conditionneed not be one that is completely deadly to the protein, butnevertheless is one that leads to a reduction in the activity of theprotein over time. Thus, a condition is denaturing if the activity ofthe protein is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or more in the presence of the condition than in itsabsence. A denaturing condition can be due to an external stress orphysical condition (e.g., agitation, temperature, time of storage,absence of a stabilizer) or can be due to the presence of a denaturingagent. For example, the denaturing condition can be caused by heat, acidor a chemical denaturant. Exemplary denaturing conditions include, butare not limited to, the presence of a strong acid or base, aconcentrated inorganic salt, an organic solvent (e.g., alcohol orchloroform), urea, high or low pH (extremes of pH), elevated temperature(e.g., heat), the presence of excipients that can be denaturing (e.g.,phenolic preservatives or detergent), and low or substantially nostabilizing agent that otherwise is required for stability of theprotein (e.g., NaCl).

As used herein, “denaturing agent” or “denaturant” refers to anysubstance, molecule or compound that causes denaturation. For example, adenaturing agent can include a strong acid or base, a concentratedinorganic salt, an organic solvent (e.g., alcohol or chloroform), apreservative, detergent or other excipient.

As used herein, “resistance to a denaturation condition” refers to anyamount of decreased reduction or elimination of a property or activityof the protein associated with or caused by denaturation. For example,denaturation is associated with or causes increased crystallization oraggregation, reduced solubility or decreased activity. Hence, resistanceto denaturation means that the protein exhibits decreased aggregation orcrystallization, increased solubility or increased or greater activity(e.g., hyaluronidase activity) when exposed to a denaturing conditioncompared to a reference protein (e.g. unmodified enzyme). The resistanceto a denaturation condition need not be absolute or permanent, but canbe achieved because the denaturation of the modifiedhyaluronan-degrading enzyme occurs more slowly than the unmodifiedenzyme in the denaturation condition such that an activity or propertyof the modified hyaluronan-degrading enzyme is achieved for longer. Forexample, a modified hyaluronan-degrading enzyme, such as a modified PH20hyaluronidase, exhibits resistance to a denaturation condition if itexhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, . . .20%, . . . 30%, . . . 40%, . . . 50%, . . . 60%, . . . , 70%, . . . 80%,. . . 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% moreresistance to denaturation in the presence of a denaturation conditionor denaturing agent than an unmodified polypeptide. In some instances, amodified polypeptide exhibits 105%, 110%, 120%, 130%, 140%, 150%, 200%,300%, 400%, 500%, or more increased resistance to denaturation comparedto an unmodified polypeptide.

As used herein, stability of a modified PH20 hyaluronidase means that itexhibits resistance to denaturation caused by a denaturation conditionor denaturing agent. A modified PH20 polypeptide exhibits stability ifit retains some activity in the presence of a denaturation condition ordenaturing agent, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or more of the original or initial hyaluronidase activity prior toexposure to the denaturing condition(s). Generally, a modified PH20hyaluronidase is stable if it retains at least 50% or more of thehyaluronidase activity under a denaturation condition compared to theabsence of the denaturation condition. Assays to assess hyaluronidaseactivity are known to one of skill in the art and described herein. Itis understood that the stability of the enzyme need not be permanent orlong term, but is manifested for a duration of time in which activity isdesired. For example, a modified PH20 hyaluronidase is stable if itexhibits an activity for at least 2 hours, 3 hours, 4 hours, 6 hours, 12hours, 24 hours, one day, two days, three days, four days, five days,six days, one week, one month, six months or one year upon exposure, orduring exposure, to one or more denaturing condition(s) or agent(s)(e.g., presence of a denaturing excipient such as a preservative). Forexample, a modified PH20 hyaluronidase is stable if it exhibits anactivity upon or during exposure to one or more denaturing condition(s)or agent(s) (e.g., presence of a denaturing excipient such as apreservative) for at least 1 month at temperatures from or from about 2°C. to 8° C., inclusive or for at least 3 days at a temperature from orfrom about 30° C. to 42° C., inclusive.

Hence, “stable” or “stability,” with reference to a formulation or aco-formulation provided herein, refers to one in which a modifiedhyaluronan-degrading enzyme, such as a modified PH20 hyaluronidase,therein is stable upon exposure to one or more denaturing condition(s)or agent(s) therein (e.g., presence of a denaturing excipient such as apreservative) for at least 1 month at temperatures from or from about 2°C. to 8° C., inclusive or for at least 3 days at a temperature from orfrom about 30° C. to 42° C., inclusive.

As used herein, “increased stability” with reference to a modified PH20hyaluronidase means that, in the presence of the same denaturing ordenaturation condition(s) (e.g., presence of a denaturing excipient suchas a preservative), the modified PH20 hyaluronidase exhibits greaterhyaluronidase activity compared to an unmodified PH20 hyaluronidase notcontaining the amino acid replacement(s). For example, a modified PH20hyaluronidase exhibits increased stability if it exhibits at least orabout at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%,200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more ofthe activity of the unmodified or reference PH20 hyaluronidase in thepresence of a denaturing or denaturation condition(s) (e.g., in thepresence of a denaturing excipient such as a preservative).

As used herein, “elevated temperatures” refers to temperatures that aregreater than room temperature or ambient temperature. Generally, anelevated temperature is a temperature that is at least, greater than, orabout 30° C., such as 30° C. to 42° C., and generally 32° C. to 37° C.or 35° C. to 37° C., inclusive.

As used herein, room temperature refers to a range generally from aboutor at to 18° C. to about or at 32° C. Those of skill in the artappreciate that room temperature varies by location and prevailingconditions. For example, room temperatures can be higher in warmerclimates such as Italy or Texas.

As used herein, recitation that proteins are “compared under the sameconditions” means that different proteins are treated identically orsubstantially identically such that any one or more conditions that caninfluence the activity or properties of a protein or agent are notvaried or not substantially varied between the test agents. For example,when the hyaluronidase activity of a modified PH20 polypeptide iscompared to an unmodified PH20 polypeptide any one or more conditionssuch as the amount or concentration of the polypeptide; presence,including amount, of excipients, carriers or other components in aformulation other than the active agent (e.g., modified PH20hyaluronidase); temperature; time of storage; storage vessel; propertiesof storage (e.g., agitation) and/or other conditions associated withexposure or use are identical or substantially identical between andamong the compared polypeptides.

As used herein, “predetermined time” refers to a time that isestablished or decided in advance. For example, the predetermined timecan be a time chosen in advance that is associated with the desiredduration of activity of a hyaluronan-degrading enzyme depending on thedesired application or use of the protein. A predetermined time can behours, days, months or years. For example, a predetermined time can beat least about or about 2 hours, 3 hours, 4 hours, five hours, sixhours, 12 hours, 24 hours, 2 days, three days, four days, five days, sixdays, one week, two weeks, three weeks, one month, six months, one yearor more.

As used herein, “storage” means that a formulation is not immediatelyadministered to a subject once prepared, but is kept for a period oftime under particular conditions (e.g., particular temperature; time,and/or form (e.g., liquid or lyophilized form)) prior to use. Forexample, a liquid formulation can be kept for days, weeks, months oryears, prior to administration to a subject under varied temperaturessuch as refrigerated (0° C. to 10° C., such as 2° to 8° C.), roomtemperature (e.g., temperature up to 32° C., such as 18° C. to about orat 32° C.), or elevated temperature (e.g., 30° C. to 42° C., such as 32°C. to 37° C. or 35° C. to 37° C.).

As used herein, an “excipient” refers to a compound in a formulation ofan active agent that does not provide the biological effect of theactive agent when administered in the absence of the active agent.Exemplary excipients include, but are not limited to, salts, buffers,stabilizers, tonicity modifiers, metals, polymers, surfactants,preservatives, amino acids and sugars.

As used herein, a stabilizing agent refers to compound added to theformulation to protect the modified PH20 polypeptide or other activeagent from degradation, if necessary, such as due to denaturationconditions to which a formulation herein is exposed when handled, storedor used. Thus, included are agents that prevent proteins fromdegradation from other components in the compositions. Exemplary of suchagents are amino acids, amino acid derivatives, amines, sugars, polyols,salts and buffers, surfactants, inhibitors or substrates and otheragents as described herein.

As used herein, an antimicrobial effectiveness test or preservativeeffectiveness test (PET) demonstrates the effectiveness of thepreservative system in a product. A product is inoculated with acontrolled quantity of specific organisms. The test then compares thelevel of microorganisms found on a control sample versus the test sampleover a period of 28 days. Generally, target markets have differing PETrequirements. For example, the PET requirements of the United StatesPharmacopoeia (USP) and the European Pharmacopoeia (EP) differ.Parameters for performing an antimicrobial effectiveness test, includingin different markets, are known to one of skill in the art as describedherein.

As used herein, an anti-microbially or anti-microbial effective amountof a preservative refers to an amount of the preservative that kills orinhibits the propagation of microbial organisms in a sample that may beintroduced from storage or use. For example, for multiple-dosecontainers, an anti-microbially effective amount of a preservativeinhibits the growth of microorganisms that may be introduced fromrepeatedly withdrawing individual doses. USP and EP (EPA and EPB) haveanti-microbial requirements that determine preservative effectiveness,and that vary in stringency. For example, an anti-microbial effectiveamount of a preservative is an amount such that at least a 1.0 log₁₀unit reduction in bacterial organisms occurs at 7 days followinginoculation in an antimicrobial preservative effectiveness test (APET).In a particular example, an anti-microbial effective amount of apreservative is an amount such that at least a 1.0 log₁₀ unit reductionin bacterial organisms occurs at 7 days following inoculation, at leasta 3.0 log₁₀ unit reduction of bacterial organisms occurs at 14 daysfollowing inoculation, at least no further increase in bacterialorganisms occurs after 28 days following inoculation, and at least noincrease in fungal organisms occurs after 7 days following inoculation.In a further example, an anti-microbial effective amount of apreservative is an amount such that at least a 1.0 log₁₀ unit reductionof bacterial organisms occurs at 24 hours following inoculation, atleast a 3.0 log₁₀ unit reduction of bacterial organisms occurs at 7 daysfollowing inoculation, no further increase in bacterial organisms occursafter 28 days following inoculation, at least a 1.0 log₁₀ unit reductionof fungal organisms occurs at 14 days following inoculation, and atleast no further increase in fungal organisms occurs after 28 daysfollowing inoculation. In an additional example, an anti-microbialeffective amount of a preservative is an amount such that at least a 2.0log₁₀ unit reduction of bacterial organisms occurs at 6 hours followinginoculation, at least a 3.0 log₁₀ unit reduction of bacterial organismsoccurs at 24 hours following inoculation, no recovery of bacterialorganisms occurs after 28 days following inoculation of the compositionwith the microbial inoculum, at least a 2.0 log₁₀ unit reduction offungal organisms occurs at 7 days following inoculation, and at least nofurther increase in fungal organisms occurs after 28 days followinginoculation.

As used herein, “preservative” refers to a naturally occurring orsynthetically or recombinantly produced substance that, when added to amolecule or protein composition, prevents microbial growth, includingbacterial or fungal growth, in the composition.

As used herein, a “phenolic preservative” refers to a preservative thatcontains one hydroxyl group attached to an aromatic carbon ring, such asa benzene ring. Exemplary phenolic preservatives, include but are notlimited to, phenol, m-cresol, p-hydroxybenzoic acid, methylparaben,ethylparaben, and propylparaben. For example, cresols, includingmeta-cresol (m-cresol), has a methyl group substituted onto the benzenering of a phenol molecule.

As used herein, a “phenophile” refers to a protein, such as a modifiedPH20 polypeptide, that exhibits stability in the presence of ananti-microbially effective amount of a preservative(s). The term“phenolphile” can be used interchangeably herein with “phenophile” andhas the same meaning. For example, a modified PH20 polypeptide that is aphenophile or phenolphile typically exhibits increased stabilitycompared to an unmodified PH20 hyaluronidase not containing the aminoacid replacement(s) when tested under the same denaturing condition(s)containing a phenolic preservative(s). For example, a modified PH20hyaluronidase exhibits at least or about at least 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%,700%, 800%, 900%, 1000% or more of the activity of the unmodified orreference PH20 hyaluronidase in the presence of a phenolicpreservative(s).

As used herein, a “thermophile” refers to a protein, such as a modifiedPH20 polypeptide, that exhibits stability under elevated temperaturesgreater than or about 30° C., such as 30° C. to 42° C., and generally32° C. to 37° C. or 35° C. to 37° C. For example, a modified PH20polypeptide that is a thermophile typically exhibits increased stabilitycompared to an unmodified PH20 hyaluronidase not containing the aminoacid replacement(s) when tested under the same elevated temperaturedenaturing condition(s). For example, a modified PH20 hyaluronidaseexhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%,1000% or more of the activity of the unmodified or reference PH20hyaluronidase under elevated temperatures.

As used herein, the term “detergent” is used interchangeably with theterm “surfactant” or “surface acting agent.” Surfactants are typicallyorganic compounds that are amphiphilic, i.e., containing bothhydrophobic groups (“tails”) and hydrophilic groups (“heads”), whichrender surfactants soluble in both organic solvents and water. Asurfactant can be classified by the presence of formally charged groupsin its head. A non-ionic surfactant has no charge groups in its head,whereas an ionic surfactant carries a net charge in its head. Azwitterionic surfactant contains a head with two oppositely chargedgroups. Some examples of common surfactants include: Anionic (based onsulfate, sulfonate or carboxylate anions): perfluorooctanoate (PFOA orPFO), perfluorooctane sulfonate (PFOS), sodium dodecyl sulfate (SDS),ammonium lauryl sulfate, and other alkyl sulfate salts, sodium laurethsulfate (also known as sodium lauryl ether sulfate, or SLES), alkylbenzene sulfonate; cationic (based on quaternary ammonium cations):cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethylammonium bromide, and other alkyltrimethylammonium salts,cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA),benzalkonium chloride (BAC), benzethonium chloride (BZT); Zwitterionic(amphoteric): dodecyl betaine; cocamidopropyl betaine; coco amphoglycinate; nonionic: alkyl poly(ethylene oxide), alkylphenolpoly(ethylene oxide), copolymers of poly(ethylene oxide) andpoly(propylene oxide) (commercially known as Poloxamers or Poloxamines),alkyl polyglucosides, including octyl glucoside, decyl maltoside, fattyalcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamideDEA, polysorbates (Tween 20, Tween 80, etc.), Triton® detergents, anddodecyl dimethylamine oxide.

As used herein, a “buffer” refers to a substance, generally a solution,that can keep its pH constant, despite the addition of strong acids orstrong bases and external influences of temperature, pressure, volume orredox potential. A buffer prevents change in the concentration ofanother chemical substance, e.g., proton donor and acceptor systems thatprevent marked changes in hydrogen ion concentration (pH). The pH valuesof all buffers are temperature and concentration dependent. The choiceof buffer to maintain a pH value or range can be empirically determinedby one of skill in the art based on the known buffering capacity ofknown buffers. Exemplary buffers include but are not limited to,bicarbonate buffer, cacodylate buffer, phosphate buffer or Tris buffer.For example, Tris buffer (tromethamine) is an amine based buffer thathas a pKa of 8.06 and has an effective pH range between 7.9 and 9.2. ForTris buffers, pH increases about 0.03 unit per ° C. temperaturedecrease, and decreases 0.03 to 0.05 unit per ten-fold dilution.

As used herein, the residues of naturally occurring α-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, a peptide refers to a polypeptide that is from 2 to 40amino acids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 1). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain). Asused herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), andadopted 37 C.F.R. §§ 1.821-1.822, abbreviations for amino acid residuesare shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic Acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic Acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or Other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence (Table 1) and modified andunusual amino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues, to an amino-terminal group such as NH₂ orto a carboxyl-terminal group such as COOH.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides.

As used herein, “non-natural amino acid” refers to an organic compoundthat has a structure similar to a natural amino acid but has beenmodified structurally to mimic the structure and reactivity of a naturalamino acid. Non-naturally occurring amino acids thus include, forexample, amino acids or analogs of amino acids other than the 20naturally-occurring amino acids and include, but are not limited to, theD-stereoisomers of amino acids. Exemplary non-natural amino acids aredescribed herein and are known to those of skill in the art.

As used herein, an isokinetic mixture is one in which the molar ratiosof amino acids has been adjusted based on their reported reaction rates(see, e.g., Ostresh et al., (1994) Biopolymers 34:1681).

As used herein, suitable conservative substitutions of amino acids areknown to those of skill in this art and can be made generally withoutaltering the biological activity of the resulting molecule. Those ofskill in the art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224). Such substitutions can be made in accordance withthose set forth in TABLE 2 as follows:

TABLE 2 Original Exemplary conservative residue substitution Ala (A)Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; ValLys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; LeuOther substitutions also are permissible and can be determinedempirically or in accord with known conservative substitutions.

As used herein, a DNA construct is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule cannot be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, “at a position corresponding to” or recitation thatnucleotides or amino acid positions “correspond to” nucleotides or aminoacid positions in a disclosed sequence, such as set forth in theSequence listing, refers to nucleotides or amino acid positionsidentified upon alignment with the disclosed sequence to maximizeidentity using a standard alignment algorithm, such as the GAPalgorithm. For purposes herein, alignment of a PH20 sequence is to theamino acid sequence set forth in any of SEQ ID NOs: 3, 7 or 32-66, andin particular SEQ ID NO:3. Hence, reference herein that a position oramino acid replacement corresponds to positions with reference to SEQ IDNO:3 also means that the position or amino acid replacement correspondsto positions with reference to any of SEQ ID NOs: 7 or 32-66, since thesequences therein are identical to the corresponding residues as setforth in SEQ ID NO:3. By aligning the sequences, one skilled in the artcan identify corresponding residues, for example, using conserved andidentical amino acid residues as guides. In general, to identifycorresponding positions, the sequences of amino acids are aligned sothat the highest order match is obtained (see, e.g.: ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAMJ Applied Math 48:1073). FIG. 2 (A-L) exemplifies exemplary alignmentsand identification of exemplary corresponding residues for replacement.

As used herein, “sequence identity” refers to the number of identical orsimilar amino acids or nucleotide bases in a comparison between a testand a reference polypeptide or polynucleotide. Sequence identity can bedetermined by sequence alignment of nucleic acid or protein sequences toidentify regions of similarity or identity. For purposes herein,sequence identity is generally determined by alignment to identifyidentical residues. Alignment can be local or global, but for purposesherein alignment is generally a global alignment where the full-lengthof each sequence is compared. Matches, mismatches and gaps can beidentified between compared sequences. Gaps are null amino acids ornucleotides inserted between the residues of aligned sequences so thatidentical or similar characters are aligned. Generally, there can beinternal and terminal gaps. Sequence identity can be determined bytaking into account gaps as the number of identical residues/length ofthe shortest sequence ×100. When using gap penalties, sequence identitycan be determined with no penalty for end gaps (e.g., terminal gaps arenot penalized). Alternatively, sequence identity can be determinedwithout taking into account gaps as the number of identicalpositions/length of the total aligned sequence ×100.

As used herein, a “global alignment” is an alignment that aligns twosequences from beginning to end, aligning each letter in each sequenceonly once. An alignment is produced, regardless of whether or not thereis similarity or identity between the sequences. For example, 50%sequence identity based on “global alignment” means that in an alignmentof the full sequence of two compared sequences each of 100 nucleotidesin length, 50% of the residues are the same. It is understood thatglobal alignment also can be used in determining sequence identity evenwhen the length of the aligned sequences is not the same. Thedifferences in the terminal ends of the sequences will be taken intoaccount in determining sequence identity, unless the “no penalty for endgaps” is selected. Generally, a global alignment is used on sequencesthat share significant similarity over most of their length. Exemplaryalgorithms for performing global alignment include the Needleman-Wunschalgorithm (Needleman et al. J. Mol. Biol. 48: 443 (1970). Exemplaryprograms for performing global alignment are publicly available andinclude the Global Sequence Alignment Tool available at the NationalCenter for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/),and the program available atdeepc2.psi.iastate.edu/aat/align/align.html.

As used herein, a “local alignment” is an alignment that aligns twosequence, but only aligns those portions of the sequences that sharesimilarity or identity. Hence, a local alignment determines ifsub-segments of one sequence are present in another sequence. If thereis no similarity, no alignment will be returned. Local alignmentalgorithms include BLAST or Smith-Waterman algorithm (Adv. Appl. Math.2: 482 (1981)). For example, 50% sequence identity based on “localalignment” means that in an alignment of the full sequence of twocompared sequences of any length, a region of similarity or identity of100 nucleotides in length has 50% of the residues that are the same inthe region of similarity or identity.

For purposes herein, sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Default parameters for the GAP program can include:(1) a unary comparison matrix (containing a value of 1 for identitiesand 0 for non identities) and the weighted comparison matrix of Gribskovet al. Nucl. Acids Res. 14: 6745 (1986), as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Whether any two nucleic acid moleculeshave nucleotide sequences or any two polypeptides have amino acidsequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical,” or other similar variations reciting a percent identity,can be determined using known computer algorithms based on local orglobal alignment (see e.g.,wikipedia.org/wiki/Sequence_alignment_software, providing links todozens of known and publicly available alignment databases andprograms). Generally, for purposes herein sequence identity isdetermined using computer algorithms based on global alignment, such asthe Needleman-Wunsch Global Sequence Alignment tool available fromNCBI/BLAST(blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign(William Pearson implementing the Huang and Miller algorithm (Adv. Appl.Math. (1991) 12:337-357)); and program from Xiaoqui Huang available atdeepc2.psi.iastate.edu/aat/align/align.html. Generally, when comparingnucleotide sequences herein, an alignment with penalty for end gaps isused. Local alignment also can be used when the sequences being comparedare substantially the same length.

Therefore, as used herein, the term “identity” represents a comparisonor alignment between a test and a reference polypeptide orpolynucleotide. In one non-limiting example, “at least 90% identical to”refers to percent identities from 90 to 100% relative to the referencepolypeptide or polynucleotide. Identity at a level of 90% or more isindicative of the fact that, assuming for exemplification purposes atest and reference polypeptide or polynucleotide length of 100 aminoacids or nucleotides are compared, no more than 10% (i.e., 10 out of100) of amino acids or nucleotides in the test polypeptide orpolynucleotide differs from that of the reference polypeptides. Similarcomparisons can be made between a test and reference polynucleotides.Such differences can be represented as point mutations randomlydistributed over the entire length of an amino acid sequence or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g., 10/100 amino acid difference (approximately 90%identity). Differences also can be due to deletions or truncations ofamino acid residues. Differences are defined as nucleic acid or aminoacid substitutions, insertions or deletions. Depending on the length ofthe compared sequences, at the level of homologies or identities aboveabout 85-90%, the result can be independent of the program and gapparameters set; such high levels of identity can be assessed readily,often without relying on software.

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wildtype form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species typicallyhave at least 80%, 90% or greater amino acid identity with a wildtypeand/or predominant form from the same species; the degree of identitydepends upon the gene and whether comparison is interspecies orintraspecies. Generally, intraspecies allelic variants have at leastabout 80%, 85%, 90% or 95% identity or greater with a wildtype and/orpredominant form, including 96%, 97%, 98%, 99% or greater identity witha wildtype and/or predominant form of a polypeptide. Reference to anallelic variant herein generally refers to variations in proteins amongmembers of the same species.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude modifications such as substitutions, deletions and insertions ofnucleotides. An allele of a gene also can be a form of a gene containinga mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human. Exemplary of species variants provided herein are primatePH20, such as, but not limited to, human, chimpanzee, macaque,cynomolgus monkey, gibbon, orangutan, or marmoset. Generally, speciesvariants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 98% sequence identity. Corresponding residues between and amongspecies variants can be determined by comparing and aligning sequencesto maximize the number of matching nucleotides or residues, for example,such that identity between the sequences is equal to or greater than95%, equal to or greater than 96%, equal to or greater than 97%, equalto or greater than 98% or equal to greater than 99%. The position ofinterest is then given the number assigned in the reference nucleic acidmolecule. Alignment can be effected manually or by eye, particularlywhere sequence identity is greater than 80%.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities, as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound can, however, be amixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

Hence, reference to a substantially purified polypeptide, such as asubstantially purified PH20 polypeptide refers to preparations of PH20proteins that are substantially free of cellular material, includespreparations of proteins in which the protein is separated from cellularcomponents of the cells from which it is isolated orrecombinantly-produced. In one embodiment, the term substantially freeof cellular material includes preparations of enzyme proteins havingless than about 30% (by dry weight) of non-enzyme proteins (alsoreferred to herein as contaminating proteins), generally less than about20% of non-enzyme proteins or 10% of non-enzyme proteins or less thanabout 5% of non-enzyme proteins. When the enzyme protein isrecombinantly produced, it also is substantially free of culture medium,i.e., culture medium represents less than about or at 20%, 10% or 5% ofthe volume of the enzyme protein preparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of enzyme proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of enzyme proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-enzymechemicals or components.

As used herein, synthetic, with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, production by recombinant means or using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce a heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well known to those of skill in the art.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells. Viral vectors include, but are not limited to,adenoviral vectors, retroviral vectors and vaccinia virus vectors.

As used herein, “operably” or “operatively linked” when referring to DNAsegments means that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiatesdownstream of the promoter and upstream of any transcribed sequences.The promoter is usually the domain to which the transcriptionalmachinery binds to initiate transcription and proceeds through thecoding segment to the terminator.

As used herein, a conjugate refers to a modified PH20 polypeptide linkeddirectly or indirectly to one or more other polypeptides or chemicalmoieties. Such conjugates include fusion proteins, those produced bychemical conjugates and those produced by any other method whereby atleast one modified PH20 polypeptide is linked, directly or indirectly toanother polypeptide or chemical moiety so long as the conjugate retainshyaluronidase activity. Exemplary of conjugates provided herein includePH20 polypeptides linked directly or indirectly to a multimerizationdomain (e.g. an Fc moiety), a toxin, a label or a drug.

As used herein, a fusion protein refers to a polypeptide encoded by anucleic acid sequence containing a coding sequence from one nucleic acidmolecule and the coding sequence from another nucleic acid molecule inwhich the coding sequences are in the same reading frame such that whenthe fusion construct is transcribed and translated in a host cell, theprotein is produced containing the two proteins. The two molecules canbe adjacent in the construct or separated by a linker polypeptide thatcontains, 1, 2, 3, or more, but typically fewer than 10, 9, 8, 7, or 6amino acids. The protein product encoded by a fusion construct isreferred to as a fusion polypeptide. Examples of fusion polypeptidesinclude Fc fusions.

As used herein, a polymer that is conjugated to a modified PH20polypeptide refers to any polymer that is covalently or otherwise stablylinked, directly or via a linker, to such polypeptide. Such polymers,typically increase serum half-life, and include, but are not limited to,sialic moieties, polyethylene glycol (PEG) moieties, dextran, and sugarand other moieties, such as for glycosylation.

As used herein, the term assessing or determining is intended to includequantitative and qualitative determination in the sense of obtaining anabsolute value for the activity of a product, and also of obtaining anindex, ratio, percentage, visual or other value indicative of the levelof the activity. Assessment can be direct or indirect.

As used herein, a “composition” refers to any mixture of two or moreproducts or compounds. It can be a solution, a suspension, liquid,powder, a paste, aqueous, non-aqueous, or any combination thereof.

As used herein, a formulation refers to a composition containing atleast one active pharmaceutical or therapeutic agent and one or moreexcipients.

As used herein, a co-formulation refers to a composition containing twoor more active or pharmaceutical or therapeutic agents and one or moreexcipients. For example, a co-formulation of a fast-acting insulin and ahyaluronan degrading enzyme contains a fast-acting insulin, a hyaluronandegrading enzyme, and one or more excipients.

As used herein, “a combination” refers to any association between two oramong more items or elements. Exemplary combinations include, but arenot limited to, two or more pharmaceutical compositions, a compositioncontaining two or more active ingredients, such as two modified PH20polypeptides; a modified PH20 polypeptide and an anticancer agent, suchas a chemotherapeutic compound; a modified PH20 polypeptide and atherapeutic agent (e.g. an insulin); a modified PH20 polypeptide and aplurality therapeutic and/or imaging agents, or any association thereof.Such combinations can be packaged as kits.

As used herein, a kit is a packaged combination, optionally, includinginstructions for use of the combination and/or other reactions andcomponents for such use.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms.

As used herein, a hyaluronan-associated disease, disorder or conditionrefers to any disease or condition in which hyaluronan levels areelevated as cause, consequence or otherwise observed in the disease orcondition. Hyaluronan-associated diseases and conditions are associatedwith elevated hyaluronan expression in a tissue or cell, increasedinterstitial fluid pressure, decreased vascular volume, and/or increasedwater content in a tissue. Hyaluronan-associated diseases, disorders orconditions can be treated by administration of a composition containinga hyaluronan degrading enzyme, such as a hyaluronidase, for example, asoluble hyaluronidase, either alone or in combination with or inaddition to another treatment and/or agent. Exemplary diseases andconditions, include, but are not limited to, hyaluronan-rich cancers,for example, tumors, including solid tumors such as late-stage cancers,metastatic cancers, undifferentiated cancers, ovarian cancer, in situcarcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer,pancreatic cancer, non-small cell lung cancer, breast cancer, coloncancer and other cancers. Exemplary hyaluronan-associated diseases andconditions also are diseases that are associated with elevatedinterstitial fluid pressure, such as diseases associated with discpressure, and edema, for example, edema caused by organ transplant,stroke, brain trauma or other injury. Exemplary hyaluronan-associateddiseases and conditions include diseases and conditions associated withelevated interstitial fluid pressure, decreased vascular volume, and/orincreased water content in a tissue, including cancers, disc pressureand edema. In one example, treatment of the hyaluronan-associatedcondition, disease or disorder includes amelioration, reduction, orother beneficial effect on one or more of increased interstitial fluidpressure (IFP), decreased vascular volume, and increased water contentin a tissue.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease. Treatment also encompasses any pharmaceuticaluse of a modified interferon and compositions provided herein.

As used herein, a pharmaceutically effective agent or therapeutic agentincludes any bioactive agent that can exhibit a therapeutic effect totreat a disease or disorder. Exemplary therapeutic agents are describedherein. Therapeutic agents include, but are not limited to, anesthetics,vasoconstrictors, dispersing agents, conventional therapeutic drugs,including small molecule drugs, including, but not limited to,bisphosphonates, and therapeutic proteins, including, but not limitedto, insulin, IgG molecules, antibodies, cytokines and coagulationfactors.

As used herein, “insulin” refers to a hormone, precursor or a syntheticor recombinant analog thereof that acts to increase glucose uptake andstorage and/or decrease endogenous glucose production. Insulin andanalogs thereof are well known to one of skill in the art, including inhuman and allelic and species variants thereof. Insulin is translated asa precursor polypeptide designated preproinsulin (110 amino acid forhuman insulin), containing a signal peptide that directs the protein tothe endoplasmic reticulum (ER) wherein the signal sequence is cleaved,resulting in proinsulin. Proinsulin is processed further to release a C-or connecting chain peptide (a 31 amino acid C-chain in human insulin).The resulting insulin contains an A-chain (21 amino acid in length inhuman insulin; set forth in SEQ ID NO:862) and a B-chain (30 amino acidin length in human insulin; set forth in SEQ ID NO:863) which arecross-linked by disulfide bonds. A fully cross-linked human insulincontains three disulfide bridges: one between position 7 of the A-chainand position 7 of the B-chain, a second between position 20 of theA-chain and position 19 of the B-chain, and a third between positions 6and 11 of the A-chain. Reference to an insulin includes monomeric andmultimeric insulins, including hexameric insulins, as well as humanizedinsulins. Exemplary insulin polypeptides are those of mammalian,including human, origin. Reference to insulin includes preproinsulin,proinsulin and insulin polypeptides in single-chain or two-chain forms,truncated forms thereof that have activity, and includes allelicvariants and species variants of human insulin, variants encoded bysplice variants, and other variants, such as insulin analogs. Anexemplary insulin is human insulin having a sequence of amino acids ofthe A- and B-chains of human insulin are set forth in SEQ ID NOs: 862and 863, respectively, and variants or analogs thereof that exhibit atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity thereto to one or both of the A-chain or B-chain andthat acts to increase glucose uptake and storage and/or decreaseendogenous glucose production. A further exemplary insulin is porcineinsulin having a sequence of amino acids for the preproinsulin as setforth in SEQ ID NO:864, whereby the A chain corresponds to amino acidresidue positions 88-108 and the B-chain correspond to amino acid, andvariants or analogs thereof that exhibit at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto toone or both of the A-chain or B-chain and that acts to increase glucoseuptake and storage and/or decrease endogenous glucose production.

As used herein, “fast-acting insulin” refers to any insulin thatexhibits peak insulin levels at or about not more than four hoursfollowing subcutaneous administration to a subject. Fast-acting insulinsinclude any insulin or any fast-acting insulin composition for acuteadministration to a diabetic subject in response to an actual,perceived, or anticipated hyperglycemic condition in the subject arisingat the time of, or within about four hours following, administration ofthe fast-acting insulin (such as a prandial hyperglycemic conditionresulting or anticipated to result from, consumption of a meal), wherebythe fast-acting insulin is able to prevent, control or ameliorate theacute hyperglycemic condition. Fast-acting insulins include recombinantinsulins and isolated insulins (also referred to as “regular” insulins)such as the insulin sold as human insulin, porcine insulins and bovineinsulins, as well as rapid acting insulin analogs (also termedfast-acting insulin analogs herein) designed to be rapid acting byvirtue of amino acid changes. Exemplary regular insulin preparationsinclude, but are not limited to, human regular insulins, such as thosesold under the trademarks Humulin® R, Novolin® R and Velosulin®, InsulinHuman, USP and Insulin Human Injection, USP, as well as acidformulations of insulin, such as, for example, Toronto Insulin, OldInsulin, and Clear Insulin, and regular pig insulins, such as Iletin II®insulin (porcine insulin). Regular insulins typically have an onset ofaction of between 30 minutes to an hour, and a peak insulin level of 2-5hours post administration.

As used herein, rapid acting insulin analogs (also called fast-actinginsulin analogs) are insulins that have a rapid onset of action. Rapidinsulins typically are insulin analogs that have been engineered, suchas by the introduction of one or more amino acid substitutions, to bemore rapid acting than regular insulins. Rapid acting insulin analogstypically have an onset of action of 10-30 minutes post injection, withpeak insulin levels observed 30-90 minutes post injection. Exemplaryrapid acting insulin analogs are analogs of human insulin containing oneor more amino acid changes in the A-chain and/or B-chain of humaninsulin set forth in SEQ ID NO:862 or 863, respectively, and thatexhibit an onset of action 10-30 minutes post injection with peakinsulin levels observed 30-90 minutes post injection. Exemplary rapidacting insulin analogs include, but are not limited to, for example,insulin lispro (e.g., Humalog® insulin), insulin aspart (e.g., NovoLog®insulin), and insulin glulisine (e.g., Apidra® insulin) the fast-actinginsulin composition sold as VIAject® and VIAtab® (see, e.g., U.S. Pat.No. 7,279,457). The amino acid sequence of exemplary rapid actinginsulin analogs have an A chain with a sequence of amino acids set forthin SEQ ID NO:862 and a B chain having a sequence of amino acids setforth in any of SEQ ID NOs:865-867. Also included are any other insulinsthat have an onset of action of 30 minutes or less and a peak levelbefore 90 minutes, typically 30-90 minutes, post injection.

As used herein, a human insulin refers to an insulin that is syntheticor recombinantly produced based upon the human polypeptide, includingallelic variants and analogs thereof.

As used herein, fast-acting human insulins or human fast-acting insulincompositions include any human insulin or composition of a human insulinthat is fast-acting, but excludes non-human insulins, such as regularpig insulin.

As used herein, the terms “basal-acting insulins,” or “basal insulins”refer to insulins administered to maintain a basal insulin level as partof an overall treatment regimen for treating a chronic condition suchdiabetes. Typically, a basal-acting insulin is formulated to maintain anapproximately steady state insulin level by the controlled release ofinsulin when administered periodically (e.g., once or twice daily).Basal-acting insulins include crystalline insulins (e.g., NPH andLente®, protamine insulin, surfen insulin), basal insulin analogs(insulin glargine, HOE 901, NovoSol Basal) and other chemicalformulations of insulin (e.g., gum arabic, lecithin or oil suspensions)that retard the absorption rate of regular insulin. As used herein, thebasal-acting insulins can include insulins that are typically understoodas long-acting (typically reaching a relatively low peak concentration,while having a maximum duration of action over about 20-30 hours) orintermediate-acting (typically causing peak insulin concentrations atabout 4-12 hours after administration).

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, therapeutic effect means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition. A therapeutically effective amount refers to the amount of acomposition, molecule or compound which results in a therapeutic effectfollowing administration to a subject.

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, a patient refers to a human subject exhibiting symptomsof a disease or disorder.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms that canbe attributed to or associated with administration of the composition ortherapeutic.

As used herein, prevention or prophylaxis refers to methods in which therisk of developing a disease or condition is reduced.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect. Hence, it is thequantity necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, unit dose form refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, a single dosage formulation refers to a formulationcontaining a single dose of therapeutic agent for direct administration.Single dosage formulations generally do not contain any preservatives.

As used herein, a multi-dose formulation refers to a formulation thatcontains multiple doses of a therapeutic agent and that can be directlyadministered to provide several single doses of the therapeutic agent.The doses can be administered over the course of minutes, hours, weeks,days or months. Multidose formulations can allow dose adjustment,dose-pooling and/or dose-splitting. Because multi-dose formulations areused over time, they generally contain one or more preservatives toprevent microbial growth.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass a therapeutic agent with a soluble PH20, such as esPH20, or anesPH20 alone, contained in the same or separate articles of packaging.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a “control” or “standard” refers to a sample that issubstantially identical to the test sample, except that it is nottreated with a test parameter, or, if it is a plasma sample, it can befrom a normal volunteer not affected with the condition of interest. Acontrol also can be an internal control. For example, a control can be asample, such as a virus, that has a known property or activity.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an” agent includes one or more agents.

As used herein, the term “or” is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. PH20 HYALURONIDASE

Provided herein are modified PH20 polypeptides. PH20 (also known assperm surface protein, sperm adhesion molecule 1 or SPAM1) is ahyaluronidase that hydrolyzes hyaluronan (also called hyaluronic acid,hyaluronate or HA) found in connective tissues such as the extracellularmatrix. Hyaluronan polymers are composed of repeating disaccharideunits, D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc),linked together via alternating β-1→4 and β-1→3 glycosidic bonds.Hyaluronan chains can reach about 25,000 disaccharide repeats or more inlength, and polymers of hyaluronan can range in size from about 5,000 to20,000,000 Da in vivo. Hyaluronan, also called hyaluronic acid orhyaluronate, is a non-sulfated glycosaminoglycan that is widelydistributed throughout connective, epithelial, and neural tissues.Hyaluronan is an essential component of the extracellular matrix and amajor constituent of the interstitial barrier. PH20 is anendo-β-N-acetyl-hexosaminidase that hydrolyzes the 131-4 glycosidic bondof hyaluronic acid into various oligosaccharide lengths such astetrasaccharides and hexasaccharides. PH20 has both hydrolytic andtransglycosidase activities. In addition to degrading hyaluronic acid,PH20 also can degrade chondroitin sulfates, such as C4-S and C6-S. PH20can exhibit hyaluronidase activity at acidic pH and neutral pH.

1. Structure

PH20 cDNA has been cloned from numerous mammalian species. ExemplaryPH20 precursor polypeptides include, but are not limited to, human (SEQID NO:6), bovine (SEQ ID NOs:15 or 17), rabbit (SEQ ID NO:23),Cynomolgus monkey (SEQ ID NO:13), guinea pig (SEQ ID NO:28), rat (SEQ IDNO:21), mouse (SEQ ID NO:19), chimpanzee (SEQ ID NO:8, SEQ ID NO:9 orSEQ ID NO:869) Rhesus monkey (SEQ ID NO:11), Fox (SEQ ID NO:30), Gibbon(SEQ ID NO:856), Marmoset (SEQ ID NO:858) or orangutan (SEQ ID NO:860)PH20 polypeptides. The mRNA transcript is typically translated togenerate a precursor protein containing a 35 amino acid signal sequenceat the N-terminus. Following transport to the ER, the signal peptide isremoved to yield a mature PH20 polypeptide. Exemplary mature PH20polypeptides include, but are not limited to, human (SEQ ID NO:7),bovine (SEQ ID NOs:16 or 18), rabbit (SEQ ID NO:24), Cynomolgus monkey(SEQ ID NO:14), guinea pig (SEQ ID NO:29), rat (SEQ ID NO:22), mouse(SEQ ID NO:20), chimpanzee (SEQ ID NO:10 or SEQ ID NO:870), Rhesusmonkey (SEQ ID NO:12), Fox (SEQ ID NO:31), Gibbon (SEQ ID NO:857),Marmoset (SEQ ID NO:859) or orangutan (SEQ ID NO:861) PH20 polypeptides.For example, the human PH20 mRNA transcript is normally translated togenerate a 509 amino acid precursor protein (SEQ ID NO:6) containing a35 amino acid signal sequence at the N-terminus (amino acid residuepositions 1-35 of SEQ ID NO:6). Thus, following transport to the ER andremoval of the signal peptide, a 474 amino acid mature polypeptide withan amino acid sequence set forth in SEQ ID NO:7 is produced. Sequencesof PH20 from ovine are also known (see e.g., SEQ ID NOs: 25-27).

In particular, human PH20 has the sequence of amino acids set forth inSEQ ID NO:6. The mature human PH20 lacking a signal sequence is setforth in SEQ ID NO:7. Allelic variants and other variants of PH20 areknown. Other sequences of PH20 have been reported. For example, a PH20variant is known as set forth in the precursor sequence set forth in SEQID NO:68 that contains an Ala at position 48 and a Trp at position 499,or the mature sequence thereof set forth in SEQ ID NO:69 containing thecorresponding differences at positions 13 and 464, respectively,compared to the sequence set forth in SEQ ID NO:7 (see e.g., Gmachl etal. (1993) FEBS Lett., 336:545-548; GenBank Accession No. AAC60607).Further, a natural variant of PH20 has been identified containing aGlutamine (Gln; Q) at position 5 as compared to the precursor sequenceof amino acids set forth in SEQ ID NO:6 (see e.g., SEQ ID NO:70, seealso Varela et al. (2011) Nature, 469:539-542). Another natural variantcontains an Alanine (Ala; A) at position 47 compared to the sequence ofamino acids set forth in SEQ ID NO:6 (as set forth in SEQ ID NO: 71) andcorresponding to position 12 compared to the sequence of amino acids setforth in SEQ ID NO: 3 or 7 (as set forth in SEQ ID NO:72).

The sequence and structure of PH20 polypeptides is highly conserved.Sequence identity between and among PH20 proteins from various speciesis about 50% to 90%. The hydrophobic N-terminal signal sequence of 35amino acids in length is generally conserved among PH20 hyaluronidasepolypeptides. PH20 hyaluronidases contain a common core hyaluronidasedomain region of about 340 amino acids in length that corresponds toamino acid residues 38-374 of the precursor human PH20 sequence setforth in SEQ ID NO:6. A mature PH20 polypeptide lacking the signalsequence and containing a contiguous sequence of amino acids having aC-terminal amino acid residue corresponding to amino acid residue 464 ofSEQ ID NO:6 (e.g., amino acid residues corresponding to positions 36-464of the amino acid sequence set forth in SEQ ID NO:6) is the minimalsequence required for hyaluronidase activity (see e.g., U.S. patentapplication Ser. No. 10/795,095, which is issued as U.S. Pat. No.7,767,429; see also U.S. Publication No. US20100143457).

Within the common hyaluronidase domain region, at least 57 amino acidsare conserved between and among species (see e.g., Arming et al. (1997)Eur. I Biochem., 247:810-814; ten Have et al. (1998) Reprod. Fertil.Dev., 10:165-72; Chowpongpang et al. (2004) Biotechnology Letters,26:1247-1252). For example, PH20 hyaluronidases contain 12 conservedcysteine residues corresponding to amino acid residue 25, 189, 203, 316,341, 346, 352, 400, 402, 408, 423 and 429 of the sequence of amino acidsof a mature PH20 lacking the signal sequence such as set forth in SEQ IDNO: 3 or 7 (corresponding to amino acid residues 60, 224, 238, 351, 376,381, 387, 435, 437, 443, 458 or 464 of full-length human PH20 set forthin SEQ ID NO:6). Cysteine residues corresponding to 25 and 316 andcysteine residues corresponding to 189 and 203 form disulfide bridges.The other cysteine residues also form disulfide bridges, are involved inposttranslational protein maturation and/or in activity modulation. Forexample, further four disulfide bonds are formed between the cysteineresidues C376 and C387; between C381 and C435; between C437 and C443;and between C458 and C464 of the polypeptide exemplified in SEQ ID NO:6(corresponding to positions C341 and C352; between C346 and C400;between C402 and C408; and between C423 and C429 of the maturepolypeptide set forth in SEQ ID NO:3 or 7, respectively).

Amino acid residues corresponding to amino acid residue D111, E113 andE249 of the sequence of amino acids set forth in SEQ ID NO: 3 or 7 areacidic residues part of the enzyme active site and are conserved betweenand among PH20 species. Amino acid residues R176, R246, R252 of thesequence of amino acids set forth in SEQ ID NO: 3 or 7 are alsoconserved between and among species and contribute to substrate bindingand/or hyaluronidase activity. Amino acid mutations D111N, E113Q, R176G,E249N and R252T result in enzymes that have no detectable enzymaticactivity or residual enzymatic activity (see e.g., Arming et al. (1997)Eur. I Biochem., 247:810-814).

The results herein confirm the requirement of PH20 amino acid residuescorresponding to positions 25, 111, 113, 176, 189, 203, 246, 249, 252,316, 341, 346, 352, 400, 402, 408, 423 and 429 of the sequence of aminoacids set forth in a mature PH20 lacking the signal sequence such as setforth in SEQ ID NO: 3 or 7 for hyaluronidase activity, since mutagenesisof these residues results in an enzyme that is not active (e.g., it isnot expressed or is inactive when expressed, see e.g., Tables 5 and 10).The exception is that amino acid replacement corresponding to R176K andC316D resulted in mutants that generated some residual hyaluronidaseactivity.

Glycosylation also is required for PH20 hyaluronidase activity based onthe recognition motif N×S or N×T. There are six N-linkedoligosaccharides at amino acid residues corresponding to positions N47,N131, N200, N219, N333 and N358 of the sequence of amino acids set forthin SEQ ID NO: 3 or 7 (corresponding to amino acid residues N82, N166,N235, N254, N368 and N393 of human PH20 set forth in SEQ ID NO: 6). Inparticular, at least N-linked glycosylation sites corresponding to aminoacid residues N200, N333 and N358 are required for secretion and/oractivity of the enzyme (see e.g., U.S. Publication No. US20100143457).For example, a PH20 polypeptide containing amino acid mutations N200A,N333A, N358A or N333A/N393A result in inactive proteins. Singlemutations of glycosylation sites N47A, N131A, N219A, N47A/N131A,N47A/N219A, N131A/N291A retain activity. The N-linked glycosylation sitecorresponding to amino acid residue N368 of human PH20 set forth in SEQID NO:6 is conserved between and among species (see e.g., Chowpongpanget al. (2004) Biotechnology Letters, 26:1247-1252). PH20 hyaluronidasesalso contains O-linked glycosylation sites. For example, human PH20 hasone O-linked oligosaccharide at the amino acid residue corresponding toamino acid T440 of the sequence of amino acids set forth in SEQ ID NO:3or 7 (corresponding to amino acid residue T475 in SEQ ID NO:6).

In addition to the catalytic sites, PH20 also contains ahyaluronan-binding site. This site is located in the Peptide 2 region,which corresponds to amino acid positions 205-235 of the precursorpolypeptide set forth in SEQ ID NO:6 and positions 170-200 of the maturepolypeptide set forth in SEQ ID NO:3 or 7. This region is highlyconserved among hyaluronidases and is similar to the heparin bindingmotif. Mutation of the arginine residue at position 176 (correspondingto the mature PH20 polypeptide set forth in SEQ ID NO:3 or 7) to aglycine results in a polypeptide with only about 1% of the hyaluronidaseactivity of the wild type polypeptide (Arming et al., (1997) Eur. IBiochem. 247:810-814).

PH20 polypeptides contain a glycosyl phosphatidylinositol (GPI) anchorattached to the C-terminus of the protein that anchors the protein tothe extracellular leaflet of the plasma membrane of cells. At leasthuman, monkey, mouse and guinea pig PH20 are strongly attached to theplasma membrane via the GPI anchor, which can be released by treatingwith phosphatidylinositol-specific phospholipase C (PI-PLC; see e.g.,Lin et al. (1994) Journal of Cell Biology, 125:1157-1163; Lin et al.(1993) Proc. Natl. Acad. Sci., 90:10071-10075). Other PH20 enzymes, suchas bovine PH20, are loosely attached to the plasma membrane and are notanchored via a phospholipase sensitive anchor. As discussed below,soluble active forms that, when expressed, are not attached to themembrane but are secreted can be generated by removal of all of aportion of the GPI anchor attachment signal site (see also U.S. Pat. No.7,767,429; U.S. Publication No. US20100143457). These include, forexample, soluble PH20 polypeptides set forth in any of SEQ ID NOs: 3 or32-66, or precursor forms thereof containing a signal sequence.

GPI-anchored proteins, for example human PH20, are translated with acleavable N-terminal signal peptide that directs the protein to theendoplasmic reticulum (ER). At the C-terminus of these proteins isanother signal sequence that directs addition of a preformed GPI-anchorto the polypeptide within the lumen of the ER. Addition of the GPIanchor occurs following cleavage of the C-terminal portion at a specificamino acid position, called the w-site (typically located approximately20-30 amino acids from the C-terminus). Although there appears to be noconsensus sequence to identify the location of the w-site, GPI anchoredproteins contain a C-terminal GPI-anchor attachment signal sequence ordomain that typically contains a predominantly hydrophobic region of8-20 amino acids, preceded by a hydrophilic spacer region of 8-12 aminoacids immediately downstream of the w-site. This hydrophilic spacerregion often is rich in charged amino acids and proline (White et al.(2000) J. Cell Sci. 113(Pt.4):721-727). There is generally a region ofapproximately 11 amino acids before the ω-1 position that ischaracterized by a low amount of predicted secondary structure, a regionaround the cleavage site (w-site), from ω-1 to ω+2 that is characterizedby the presence of small side chain residues, the spacer region betweenpositions ω+3 and ω+9, and a hydrophobic tail from ω+10 to theC-terminal end (Pierleoni et al., (2008) BMC Bioinformatics 9:392).

Although there is no GPI-anchor attachment signal consensus sequence,various in silico methods and algorithms have been developed that can beused to identify such sequences in polypeptides (see, e.g., Udenfriendet al. (1995)Methods Enzymol. 250:571-582; Eisenhaber et al. (1999) J.Mol. Chem. 292: 741-758; Kronegg and Buloz, (1999),“Detection/prediction of GPI cleavage site (GPI-anchor) in a protein(DGPI),” 129.194.185.165/dgpi/; Fankhauser et al. (2005) Bioinformatics21:1846-1852; Omaetxebarria et al. (2007) Proteomics 7:1951-1960;Pierleoni et al. (2008) BMC Bioinformatics 9:392), including those thatare readily available on bioinformatic websites, such as the ExPASyProteomics tools site (expasy.ch/tools/). Thus, one of skill in the artcan determine whether a PH20 polypeptide likely contains a GPI-anchorattachment signal sequence, and, therefore, whether the PH20 polypeptideis a GPI-anchored protein.

The covalent attachment of a GPI-anchor to the C-terminus of human PH20and, therefore, the membrane-bound nature of PH20, has been confirmedusing phosphatidylinositol-specific phospholipase C (PI-PLC) hydrolysisstudies (see e.g., Lin et al., (1994) J. Biol. Chem. 125:1157-1163).Phosphatidylinositol-specific phospholipase C (PI-PLC) and D (PI-PLD)hydrolyze the GPI anchor, releasing the PH20 polypeptide from the cellmembrane. The prior art literature reports that a ω-site cleavage siteof human PH20 is identified between Ser-490 and Ala-491 and for monkeyPH20 is identified between Ser491 and Thr492 (Lin et al. (1993) Proc.Natl. Acad. Sci, (1993) 90:10071-10075). Thus, the literature reportsthat a GPI-anchor attachment signal sequence of human PH20 is located atamino acid positions 491-509 of the precursor polypeptide set forth inSEQ ID NO:6, and the ω-site is amino acid position 490. Thus, in thismodeling of human PH20, amino acids 491-509 are cleaved followingtransport to the ER and a GPI anchor is covalently attached to theserine residue at position 490.

2. Function

PH20 is normally expressed in sperm from a single testis-specific gene.PH20 is a sperm-associated protein involved in fertilization. PH20 isnormally localized on the sperm surface, and in the lysosome-derivedacrosome, where it is bound to the inner acrosomal membrane. PH20 ismultifunctional and exhibits hyaluronidase activity, hyaluronan(HA)-mediated cell-signaling activity, and acts as a sperm receptor forthe zona pellucida surrounding the oocyte when present on acrosomereacted (AR) sperm. For example, PH20 is naturally involved in sperm-eggadhesion and aids penetration by sperm of the layer of cumulus cells bydigesting hyaluronic acid. In addition to being a hyaluronidase, PH20also appears to be a receptor for HA-induced cell signaling, and areceptor for the zona pellucida surrounding the oocyte. Due to the roleof PH20 in fertilization, PH20 can be used as an antigen forimmunocontraception.

PH20 is a neutral active hyaluronidase, although it can exhibitacid-active activity in some cases. The hyaluronidase activity of PH20is exhibited by the plasma membrane- and inner acrosomalmembrane-associated PH20. The plasma membrane PH20 exhibitshyaluronidase activity only at neutral pH, while the inner acrosomalmembrane-associated PH20 exhibits acid-active enzyme activity. Thestructural basis for these differences is due to the presence of twocatalytic sites in PH20. A first catalytic site is designated thePeptide 1 region, corresponding to amino acid residues 142-172 of SEQ IDNO:6, which is involved in enzyme activity of PH20 at neutral pH. Asecond catalytic site is designated the peptide 3 region, correspondingto amino acid residues 277-297 of SEQ ID NO:6, which is involved inenzyme activity at lower pH. A change in the structure of the inneracrosomal membrane-associated PH20 occurs after the acrosome reaction,whereby PH20 is endoproteolytically cleaved but held together bydisulfide bonds. The result of the endoproteolysis is that the peptide 3region is activated and can thus effect neutral and acid-activity toPH20 (see e.g., Cherr et al. (2001) Matrix Biology, 20:515-525. Also,after the acrosome reaction, lower molecular weight forms are generatedby release from the inner acrosomal membrane (e.g., a 53 kDa solubleform of PH20 is generated in monkey). The lower molecular weight form(s)also is acid active.

The hyaluronidase activity of PH20 accounts for the spreading activityobserved in animal testes extracts that have been used clinically fordecades to increase the dispersion and absorption of drugs (see e.g.,Bookbinder et al. (2006) J. Controlled Release, 114:230-241). Forexample, pharmaceutical preparations containing hyaluronidase weredeveloped as fractionated extracts from bovine testes for therapeuticuse as spreading agents and in other applications (Schwartzman (1951) J.Pediat., 39:491-502). Original bovine testicular extract preparationsincluded, for example, extracts sold under the trademarks Wydase®,Hylase®, “Dessau,” Neopermease®, Alidase® and Hyazyme®. It is now knownthat the spreading activity of testicular extract preparations are dueto PH20 hyaluronidase activity. For example, in 2001 a spermhyaluronidase in bull was identified as the hyaluronidase PH20(Lalancette et al. (2001) Biol. Reprod., 65:628-36). By catalyzing thehydrolysis of hyaluronic acid, PH20 hyaluronidase lowers the viscosityof hyaluronic acid, thereby increasing tissue permeability. Hence,soluble forms of PH20 are used as a spreading or dispersing agent inconjunction with other agents, drug and proteins to enhance theirdispersion and delivery, and to improve the pharmacokinetic andpharmacodynamic profile of the coadministered agent, drug or protein(see e.g., U.S. Pat. No. 7,767,429; Bookbinder et al. (2006) J.Controlled Release, 114:230-241).

3. Soluble PH20 Polypeptides

PH20 can exist in membrane-bound or membrane-associated form, or can besecreted into the media when expressed from cells, and thereby can existin soluble form. Soluble PH20 can be detected and discriminated frominsoluble, membrane-bound PH20 using methods well known in the art,including, but not limited to, those using a Triton® X-114 detergentassay. In this assay, soluble PH20 hyaluronidases partition into theaqueous phase of a Triton® X-114 detergent solution warmed to 37° C.(Bordier et al., (1981) J. Biol. Chem., 256:1604-7) whilemembrane-anchored PH20 hyaluronidases partition into the detergent richphase. Thus, in addition to using algorithms to assess whether a PH20polypeptide is naturally GPI-anchored and hence membrane-bound,solubility experiments also can be performed.

Soluble PH20 enzymes include hyaluronidases that contain a GPI-anchorattachment signal sequence, but that are loosely attached to themembrane such that they do not contain a phospholipase sensitive anchor.For example, soluble PH20 polypeptides include ovine or bovine PH20.Various forms of such soluble PH20 hyaluronidases have been prepared andapproved for therapeutic use in subjects, including humans. For example,animal-derived hyaluronidase preparations include Vitrase® hyaluronidase(ISTA Pharmaceuticals), which is a purified ovine testicularhyaluronidase, and Amphadase® hyaluronidase (Amphastar Pharmaceuticals),which is a bovine testicular hyaluronidase. Soluble PH20 enzymes alsoinclude truncated forms of non-human or human membrane-associated PH20hyaluronidases that lack one or more amino acid residues of aglycosylphosphatidylinositol (GPI) anchor attachment signal sequence andthat retain hyaluronidase activity (see e.g., U.S. Pat. No. 7,767,429;U.S. Publication No. US20100143457). Thus, instead of having aGPI-anchor covalently attached to the C-terminus of the protein in theER and being anchored to the extracellular leaflet of the plasmamembrane, these polypeptides are secreted when expressed from cells andare soluble. In instances where the soluble hyaluronan degrading enzymeretains a portion of the GPI anchor attachment signal sequence, 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more amino acid residues in the GPI-anchorattachment signal sequence can be retained, provided the polypeptide issoluble (i.e., secreted when expressed from cells) and active.

Exemplary soluble hyaluronidases that are C-terminally truncated andlack all or a portion of the GPI anchor attachment signal sequenceinclude, but are not limited to, PH20 polypeptides of primate origin,such as, for example, human and chimpanzee PH20 polypeptides. Forexample, soluble PH20 polypeptides can be made by C-terminal truncationof a polypeptide set forth in SEQ ID NO:7, 10, 12, 14, 69, 72, 857, 859,861 or 870 or variants thereof that exhibit at least 80%, 85%, 90%, 95%or more sequence identity to any of SEQ ID NO: 7, 10, 12, 14, 69, 72,857, 859, 861 or 870, wherein the resulting polypeptide is active,soluble and lacks all or a portion of amino acid residues from theGPI-anchor attachment signal sequence.

Exemplary soluble PH20 polypeptides are C-terminal truncated human PH20polypeptides that are mature (lacking a signal sequence), soluble andexhibit neutral activity, and that contain a contiguous sequence ofamino acids set forth in SEQ ID NO:6 or SEQ ID NO:7 that minimally has aC-terminal truncated amino acid residue at or after amino acid residue464 of the sequence of amino acids set forth in SEQ ID NO:6. Forexample, soluble PH20 polypeptides include C-terminal truncatedpolypeptides that minimally contain a contiguous sequence of amino acids36-464 of SEQ ID NO:6, or includes a sequence of amino acids that has atleast 85%, for example at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% sequence identity to a contiguous sequence ofamino acids that has a C-terminal amino acid residue after amino acid464 of SEQ ID NO:6 and retains hyaluronidase activity. ExemplaryC-terminally truncated human PH20 polypeptides are mature polypeptides(lacking a signal sequence) that include a contiguous sequence of aminoacids set forth in SEQ ID NO:6 with a C-terminal residue after 464 suchas after amino acid position 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500of the sequence of amino acids set forth in SEQ ID NO:6, or a variantthereof that exhibits at least 85% sequence identity, such as at least86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequenceidentity thereto and retains hyaluronidase activity. For example,exemplary C-terminal PH20 polypeptides have a sequence of amino acids 36to 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499 or 500 of the sequence of amino acidsset forth in SEQ ID NO:6, or a variant thereof that exhibits at least85% sequence identity, such as at least 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity thereto and retainshyaluronidase activity. Soluble PH20 polypeptides include any that hasthe sequence of amino acids set forth in SEQ ID NOs: 3 or 32-66 or asequence of amino acids that exhibits at least 85% sequence identity,such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% sequence identity to the sequence of amino acids set forth inany of SEQ ID NOs: 3 or 32-66.

In particular, a soluble human PH20 polypeptide is a polypeptide that istruncated after amino acid 482 of the sequence set forth in SEQ ID NO:6.Such a polypeptide can be generated from a nucleic acid moleculecontaining a signal sequence and encoding amino acids 36-482, forexample, as set forth in SEQ ID NO:1 (containing an IgG kappa signalsequence) or SEQ ID NO:67 (containing the native signal sequence). Posttranslational processing removes the signal sequence, leaving a 447amino acid soluble recombinant human PH20 (SEQ ID NO:3). A productproduced upon expression of a vector set forth in SEQ ID NO:4 or 5, andcontaining a nucleic acid molecule set forth in SEQ ID NO:67, results ina secreted product, designated rHuPH20, in the culture medium thatexhibits heterogeneity at the C-terminus such that the product includesa mixture of species that can include any one or more of SEQ ID NOs: 3and 44-48 in various abundance. Typically, rHuPH20 is produced in cellsthat facilitate correct N-glycosylation to retain activity, such asmammalian cells, for example CHO cells (e.g., DG44 CHO cells). Hylenex®hyaluronidase (Halozyme) is a human recombinant hyaluronidase producedby genetically engineered Chinese Hamster Ovary (CHO) cells containingnucleic acid encoding a truncated human PH20 polypeptide (designatedrHuPH20).

C. MODIFIED PH20 POLYPEPTIDES

Provided herein are modified or variant PH20 polypeptides. The modifiedPH20 polypeptides provided herein exhibit altered activities orproperties compared to a wildtype, native or reference PH20 polypeptide.Included among the modified PH20 polypeptides provided herein are PH20polypeptide that are active mutants, whereby the polypeptides exhibit atleast 40% of the hyaluronidase activity of the corresponding PH20polypeptide not containing the amino acid modification (e.g., amino acidreplacement). In particular, provided herein are PH20 polypeptides thatexhibit hyaluronidase activity and that exhibit increased stabilitycompared to the PH20 not containing the amino acid modification. Alsoprovided are modified PH20 polypeptides that are inactive, and that canbe used, for example, as antigens in contraception vaccines.

The modifications can be a single amino acid modification, such assingle amino acid replacements (substitutions), insertions or deletions,or multiple amino acid modifications, such as multiple amino acidreplacements, insertions or deletions. Exemplary modifications are aminoacid replacements, including single or multiple amino acid replacements.The amino acid replacement can be a conservative substitution, such asset forth in Table 2, or a non-conservative substitution, such as anydescribed herein. Modified PH20 polypeptides provided herein can containat least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more modified positions compared to the PH20 polypeptidenot containing the modification.

The modifications described herein can be in any PH20 polypeptide,including, including precursor, mature, or C-terminal truncated forms,so long as the modified form exhibits hyaluronidase activity. Forexample, the PH20 polypeptides contain modifications compared to awildtype, native or reference PH20 polypeptide set forth in any of SEQID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870, or in a polypeptide thathas a sequence of amino acids that is at least 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any of SEQ ID NOs: 3, 6-66, 68-72, 856-861, 869 or 870. Forexample, the modifications are made in a human PH20 polypeptide havingthe sequence of amino acids including or set forth in SEQ ID NO:7, SEQID NO:69 or SEQ ID NO:72; a bovine PH20 polypeptide having a sequence ofamino acids including or set forth in SEQ ID NOs:16 or 18; a rabbit PH20polypeptide having a sequence of amino acids including or set forth inSEQ ID NO:24; a Cynomolgus monkey PH20 polypeptide having a sequence ofamino acids including or set forth in SEQ ID NO:14; a guinea pig PH20polypeptide having a sequence of amino acids including or set forth inSEQ ID NO:29; a rat PH20 polypeptide having a sequence of amino acidsincluding or set forth in SEQ ID NO:22; a mouse PH20 polypeptide havinga sequence of amino acids including or set forth in SEQ ID NO:20; achimpanzee PH20 polypeptide having a sequence of amino acids includingor set forth in SEQ ID NO:10 or 870; a Rhesus monkey PH20 polypeptidehaving a sequence of amino acids including or set forth in SEQ ID NO:12;a Fox PH20 polypeptide having a sequence of amino acids including or setforth in SEQ ID NO:31; a Gibbon PH20 polypeptide having a sequence ofamino acids including or set forth in SEQ ID NO:857; a Marmoset PH20polypeptide having a sequence of amino acids including or set forth inSEQ ID NO: 859; an Orangutan PH20 polypeptide having a sequence of aminoacids including or set forth in SEQ ID NO:861; or a sheep PH20polypeptide having a sequence of amino acids including or set forth inany of SEQ ID NOs: 25-27; or in sequence variants or truncated variantsthat exhibit at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toany of SEQ ID NOs: 7, 10, 12, 14, 16, 18, 20, 22, 24-27, 29, 31, 69, 72,857, 859, 861 or 870.

In particular, provided herein are PH20 polypeptides that containmodifications compared to a PH20 polypeptide set forth in SEQ ID NO: 3,7, 32-66, 69 or 72, or a polypeptide that has a sequence of amino acidsthat is at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs:3, 7, 32-66, 69 or 72. For example, the modifications provided hereinalso can be made in a PH20 polypeptide set forth as SEQ ID NO: 10, 12,14, 24, 857, 859, 861 or 870.

In particular, provided herein are modified soluble PH20 polypeptidesthat are PH20 polypeptides containing a modification provided herein,and that when expressed from cells are secreted into the media as asoluble protein. For example, the modifications are made in a solublePH20 polypeptide that is C-terminally truncated within or near theC-terminus portion containing the GPI-anchor signal sequence of a PH20polypeptide that contains a GPI-anchor signal sequence. The C-terminaltruncation can be a truncation or deletion of 8 contiguous amino acidsat the C-terminus, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids at theC-terminus, so long as the resulting C-terminally truncated polypeptideexhibits hyaluronidase activity and is secreted from cells (e.g., intothe media) when expressed. In some examples, the modifications providedherein are made in a soluble PH20 polypeptide that is a C-terminallytruncated polypeptide of SEQ ID NO:7, 10, 12, 14, 69, 72, 857, 859, 861or 870 or a variant thereof that exhibits at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to any of SEQ ID NOs: 7, 10, 12, 14, 69, 72, 857,859, 861 or 870. In particular, the modifications provided herein aremade in a soluble or C-terminally truncated human PH20 polypeptidehaving the sequence of amino acids set forth in SEQ ID NOs: 3 or 32-66or a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequenceidentity to the sequence of amino acids set forth in any of SEQ ID NOs:3 or 32-66. For example, modified PH20 polypeptides provided hereincontain amino acid replacements or substitutions, additions ordeletions, truncations or combinations thereof with reference to thePH20 polypeptide set forth in SEQ ID NO:3.

Modifications also can be made in the corresponding precursor formcontaining a signal peptide of any of SEQ ID NOs: 3, 7, 10, 12, 14, 16,18, 20, 22, 24-27, 29, 31, 32-66, 69, 72, 857, 859, 861 or 870. Forexample, modifications provided herein can be made in a precursor formset forth in any of SEQ ID NOs: 2, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23,28, 30, 856, 858, 860 or 869 or in a variant thereof that exhibits atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:2, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 28, 30, 856, 858, 860 or 869.

In examples of modified PH20 polypeptides provided herein, the modifiedPH20 polypeptide does not contain the sequence of amino acids set forthin any of SEQ ID NOs: 3-66, 68-72, 856-861, 869 or 870. Typically, themodified PH20 polypeptide is a human PH20 polypeptide, and does notcontain the sequence of amino acids set forth in any of SEQ ID NOs:8-31, 856-861, 869 or 870.

Generally, any modification, such as amino acid replacement, deletion orsubstitution, can be made in a PH20 polypeptide, with the proviso thatthe modification is not an amino acid replacement where the onlymodification is a single amino acid replacement that is V12A, N47A,D111N, E113Q, N131A, R176G, N200A, N219A, E249Q, R252T, N333A or N358A.Also, where the modified PH20 polypeptide contains only two amino acidreplacements, the amino acid replacements are not P13A/L464W,N47A/N131A, N47A/N219A, N131A/N219A or N333A/N358A. In a furtherexample, where the modified PH20 polypeptide contains only three aminoacid replacements, the amino acid replacements are not N47A/N131A/N219A.Exemplary modifications provided herein are described in detail below.

For purposes herein, reference to positions and amino acids formodification herein, including amino acid replacement or replacements,are with reference to the PH20 polypeptide set forth in SEQ ID NO:3. Itis within the level of one of skill in the art to make any of themodifications provided herein in another PH20 polypeptide by identifyingthe corresponding amino acid residue in another PH20 polypeptide, suchas any set forth in SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24-27, 28, 29, 30, 31, 32-66, 68-72, 856,857, 858, 859, 860, 861, 869 or 870 or a variant thereof that exhibitsat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ IDNOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24-27, 28, 29, 30, 31, 32-66, 68-72, 856, 857, 858, 859, 860, 861, 869or 870. Corresponding positions in another PH20 polypeptide can beidentified by alignment of the PH20 polypeptide with the reference tothe PH20 polypeptide set forth in SEQ ID NO:3. For example, FIG. 2 (A-L)depicts alignment of exemplary PH20 polypeptides with SEQ ID NO:3, andidentification of exemplary corresponding positions. Also, since SEQ IDNOs: 3, 7, 32-66, 69 and 72 are all forms of a mature human PH20 with adifferent C-terminal amino acid residue, the numbering of amino acidresidues in any of SEQ ID NOs: 7, 32-66, 69 and 72 is the same as SEQ IDNO:3, and hence the corresponding residues of each are identical to thatset forth in SEQ ID NO:3 (see e.g., FIG. 1 ). Further, SEQ ID NOS setforth in any of SEQ ID NOs: 2, 6, 70 or 71 are precursor forms thereofthat differ by only the presence of a signal sequence. For purposes ofmodification (e.g., amino acid replacement), the corresponding aminoacid residue can be any amino acid residue, and need not be identical tothe residue set forth in SEQ ID NO:3. Typically, the corresponding aminoacid residue identified by alignment with residues in SEQ ID NO:3 is anamino acid residue that is identical to SEQ ID NO:3, or is aconservative or semi-conservative amino acid residue thereto (see e.g.,FIGS. 2A-2L). It is also understood that the exemplary replacementsprovided herein can be made at the corresponding residue in a PH20polypeptide, so long as the replacement is different than exists in theunmodified form of the PH20 polypeptide. Based on this description andthe description elsewhere herein, it is within the level of one of skillin the art to generate a modified PH20 polypeptide containing any one ormore of the described mutation, and test each for a property or activityas described herein.

Modifications in a PH20 polypeptide also can be made to a PH20polypeptide that also contains other modifications, includingmodifications of the primary sequence and modifications not in theprimary sequence of the polypeptide. For example, modificationsdescribed herein can be in a PH20 polypeptide that is a fusionpolypeptide or chimeric polypeptide. The modified PH20 polypeptidesprovided herein also include polypeptides that are conjugated to apolymer, such as a PEG reagent.

Also provided herein are nucleic acid molecules that encode any of themodified PH20 polypeptides provided herein. In particular examples, thenucleic acid sequence can be codon optimized, for example, to increaseexpression levels of the encoded sequence. The particular codon usage isdependent on the host organism in which the modified polypeptide isexpressed. One of skill in the art is familiar with optimal codons forexpression in mammalian or human cells, bacteria or yeast, including forexample E. coli or Saccharomyces cerevisiae. For example, codon usageinformation is available from the Codon Usage Database available atkazusa.or.jp.codon (see Richmond (2000) Genome Biology, 1:reports241 fora description of the database). See also, Forsburg (1994) Yeast,10:1045-1047; Brown et al. (1991) Nucleic Acids Research, 19:4298; Sharpet al. (1988) Nucleic Acids Res., 12:8207-8211; Sharp et al. (1991)Yeast, 657-78). In some examples, the encoding nucleic acid moleculesalso can be modified to contain a heterologous signal sequence to alter(e.g., increased) expression and secretion of the polypeptide. Exemplaryof a heterologous signal sequence is a nucleic acid encoding the IgGkappa signal sequence (set forth in SEQ ID NO:868).

The modified polypeptides and encoding nucleic acid molecules providedherein can be produced by standard recombinant DNA techniques known toone of skill in the art. Any method known in the art to effect mutationof any one or more amino acids in a target protein can be employed.Methods include standard site-directed or random mutagenesis of encodingnucleic acid molecules, or solid phase polypeptide synthesis methods.For example, nucleic acid molecules encoding a PH20 polypeptide can besubjected to mutagenesis, such as random mutagenesis of the encodingnucleic acid, error-prone PCR, site-directed mutagenesis, overlap PCR,gene shuffling, or other recombinant methods. The nucleic acid encodingthe polypeptides can then be introduced into a host cell to be expressedheterologously. Hence, also provided herein are nucleic acid moleculesencoding any of the modified polypeptides provided herein. In someexamples, the modified PH20 polypeptides are produced synthetically,such as using solid phase or solutions phase peptide synthesis.

In the subsections below, exemplary modified PH20 polypeptide exhibitingaltered properties and activities, and encoding nucleic acid molecules,provided herein are described.

1. Active Mutants

Provided herein are modified PH20 polypeptides that contain one or moreamino acid replacements in a PH20 polypeptide and that exhibithyaluronidase activity. The modified PH20 polypeptides can exhibit 40%to 5000% of the hyaluronidase activity of a wildtype or reference PH20polypeptide, such as the polypeptide set forth in SEQ ID NOs: 3 or 7.For example, modified PH20 polypeptides provided herein exhibit at least40% of the hyaluronidase activity, such as at least 50%, 60%, 70%, 80%,90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000% or more of thehyaluronidase activity of a wildtype or reference PH20 polypeptide, suchas the corresponding polypeptide not containing the amino acidmodification (e.g., amino acid replacement), for example, a polypeptideset forth in SEQ ID NO:3 or 7. For example, exemplary positions that canbe modified, for example by amino acid replacement or substitution,include, but are not limited to, any of positions corresponding toposition 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 24,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 58, 59, 60, 61, 63, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 89,90, 91, 92, 93, 94, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107, 108,110, 114, 117, 118, 119, 120, 122, 124, 125, 127, 128, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 192, 193, 195,196, 197, 198, 200, 202, 204, 205, 206, 208, 209, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 224, 226, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 242, 245, 247, 248, 251, 253, 255,256, 257, 258, 259, 260, 261, 263, 264, 265, 266, 267, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 297, 298, 300, 301, 302, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,320, 321, 323, 324, 325, 326, 327, 328, 331, 334, 335, 338, 339, 342,343, 347, 348, 349, 351, 353, 356, 357, 358, 359, 360, 361, 367, 368,369, 371, 373, 374, 375, 376, 377, 378, 379, 380, 381, 383, 385, 387,388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 401, 403, 404,405, 406, 407, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419,420, 421, 422, 425, 426, 427, 428, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446 or 447 with reference toamino acid positions set forth in SEQ ID NO:3. Typically, the amino acidresidue that is modified (e.g., replaced with another amino acid) at theposition corresponding to any of the above positions in a PH20polypeptide is an identical residue, a conservative residue or asemi-conservative amino acid residue to the amino acid residue set forthin SEQ ID NO:3.

To retain hyaluronidase activity, modifications typically are not madeat those positions that are less tolerant to change or required forhyaluronidase activity. For example, generally modifications are notmade at a position corresponding to position 7, 16, 17, 18, 19, 21, 25,53, 55, 56, 57, 62, 64, 76, 78, 80, 88, 95, 100, 101, 109, 111, 112,113, 115, 116, 121, 123, 126, 129, 185, 187, 188, 189, 190, 191, 194,199, 201, 203, 207, 210, 223, 225, 227, 228, 229, 241, 243, 244, 246,249, 250, 252, 254, 262, 268, 295, 296, 299, 303, 319, 322, 329, 330,332, 333, 336, 337, 340, 341, 344, 345, 346, 350, 352, 354, 355, 362,363, 364, 365, 366, 370, 372, 382, 384, 386, 390, 400, 402, 408, 423,424, 429, 430, with reference to amino acid positions set forth in SEQID NO:3. Also, in examples where modifications are made at any ofpositions 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 27,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 54, 58, 59, 60, 61, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 94, 96, 98, 99,102, 103, 104, 105, 106, 107, 108, 110, 114, 117, 118, 119, 122, 124,125, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 143,144, 145, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 161, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 186, 192, 193, 195, 197, 198, 200,202, 204, 206, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 224, 226, 230, 231, 232, 233, 234, 235, 236, 238, 239,240, 242, 245, 247, 248, 251, 253, 255, 256, 257, 258, 260, 261, 263,264, 265, 266, 267, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279,280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,297, 298, 300, 301, 302, 304, 305, 306, 307, 308, 310, 311, 312, 313,314, 315, 316, 317, 318, 320, 321, 323, 324, 325, 326, 327, 331, 334,335, 338, 339, 342, 343, 347, 348, 349, 351, 353, 356, 357, 358, 359,360, 361, 367, 368, 369, 371, 373, 374, 375, 376, 377, 378, 379, 380,381, 383, 385, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398,399, 401, 403, 404, 405, 406, 410, 411, 412, 413, 414, 415, 416, 417,419, 420, 422, 425, 426, 427, 428, 431, 432, 434, 437, 438, 439, 440,441, 442, 443, 444, or 447 with reference to amino acid positions setforth in SEQ ID NO:3, the modification(s) is/are not the correspondingamino acid replacement(s) set forth in Table 5 or 10 herein, which areamino acid replacements that result in an inactive polypeptide. Forexample, if the modification is a modification at a positioncorresponding to position 2 with reference to SEQ ID NO:3, themodification is not replacement to a histidine (H), lysine (K),tryptophan (W) or tyrosine (Y).

Exemplary amino acid replacements at any of the above correspondingpositions are set forth in Table 3. Reference to the corresponding aminoacid position in Table 3 is with reference to positions set forth in SEQID NO:3. It is understood that the replacements can be made in thecorresponding position in another PH20 polypeptide by alignmenttherewith with the sequence set forth in SEQ ID NO:3 (see e.g., FIGS. 1and 2 ), whereby the corresponding position is the aligned position. Inparticular examples, the amino acid replacement(s) can be at thecorresponding position in a PH20 polypeptide as set forth in any of SEQID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereofhaving at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto, so long as the resulting modified PH20 polypeptideexhibits at least 40% of the hyaluronidase activity of the correspondingPH20 polypeptide not containing the amino acid replacement. Inparticular, the replacement(s) can be in a corresponding position in ahuman PH20 polypeptide, for example, any set forth in any of SEQ ID NOs:3, 7, 32-66, 69 or 72, or a variant thereof that exhibits at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toany of SEQ ID NOs: 3, 7, 32-66, 69 or 72. In one example, any one ormore of the replacements are in SEQ ID NO:3, so long as the resultingmodified PH20 polypeptide exhibits at least 40% of the hyaluronidaseactivity of the PH20 polypeptide set forth in SEO ID NO:3.

TABLE 3 Active Mutants Corresponding Corresponding CorrespondingPosition Replacement Position Replacement Position Replacement 1A C E F G H K N P 2 A C G I L P Q S T 3 E H L Y Q R S T V W V 4A I S T V 5 H 6 A H K L N Q R 7 M 8 I L M P  9 K L Q R S V 10D E G H N Q R S W 11 D G H K S 12 A E I K L N R S T 13 H S T Y 14D I M V 15 A M V 20 S 22 H M T Y 23 D 24 A E G H I K L M N 26A E G H I K M P Q 27 A D E F H I K L P R T V Y R S T V W Y Q R S T W 28A D E F I L M N P 29 A E G H I K L M P 30 A F G H K L M P R S T V WR S T V W Q R S T V W 31 A C G H I K L P R S 32 A C F G H K L M N 33G M P Q R S T W T V W Y Q R S T V W Y 34 A E H K Q R W 35 F H L Q T V Y36 A D G H K L N R T 37 F I K M P R W V 38 Y 39 A L N Q R T Y 40 L W 41A C D E G H N T V 42 A W 43 N T 44 E 45 I K 46 A C E F H L M N R 47A D F G H K M Q 48 F G H I K M N Q R S T V Y R S T W Y S V Y 49I K R S V 50 A C D E H L M Q R 51 A N R S S V Y 52 N P Q R S T 54A F N Q S V 58 C G H I K L N P Q R S W Y 59 Q N 60 K 61 F I M V 63A H I K L M N R S 65 R 66 H R T V W 67 F L R V Y 68 E G H K L P Q R S 69A C E F G I L M P T R T W Y 70 A C F G H K L N P 71 A D G H L M N Q 72A D E H K L M Q R S T V Y R S R S Y 73 A C D G H K L M Q 74A C E F G H K L M 75 A C F H L M N Q R S T W N P R S V W R S T Y 77 H K79 L T V 81 P 82 A E G H I L M N Q R S T V 83 F G H K L N Q R S 84D E F G H I L M N 85 V T V P Q R T W Y 86 A D E F G H I K L 87A C E G H I L M P 89 C K M P R W M N P R S T V W Q R S T V Y 90A E G H I K L N Q 91 A Q R 92 C H L M T V R S T W 93 D E F G H I L M N94 A C D E F H L M N 96 D L V P Q R S T V Q R S T 97 A C D E F G I L N P98 A C D E H I L M Q 99 A R S Q R S W Y R S V W 102 A C E G H K L M NQ R S T W 103 N 104 A C G I K M R S T 105 A C G H I P Q R S T W V 106 V107 F I L 108 G 110 V 114 A G H M S 117 D 118 H K L M N Q V 119 F P Q Y120 D F G H I L N P R 122 M S T V W Y 124 H L R 125 A H R S 127A E G H L M N Q R S T V W 128 A C G I K L Q R S 130 I R 131C E F G H I L M Q W R S T V Y 132 A C E F H I K L N 133 I 134 L T VQ S T V Y 135 A C D F G H K L N 136 A C D F H I M N Q 137A C I T A C H I L Q R S W Y R S T W M N R S W Y 139 A C D E F G H K L140 A C D F G H I K L 141 A D E F G H L M M R S T V M R V W YQ R S T V W Y 142 C D E G H I K L M 143 C E G I K L N V 144 R T WN P Q R S T 145 A C D E G H L M N 146 A C E G H I K N P 147A C D F G I L M P P R Q R S T V Y Q R S V W Y 148 C F G H I K L Q R S149 C G K L M Q R S T 150 A C D E F G I L N T V W Y V P R S W Y 151A C G H K L M N Q 152 A C F I M R T V W 153 I L S R S T V W Y Y 154I R T V 155 A C D F G H K L M 156 A C D G I L M Q R R S T V W S T V W157 W 158 A F G H L Q S 159 A D E G H L M N Q R S V 160C F G H I K L M N 161 A C D E R S V 162 A D E G H L M P Q R S W V YQ R S V W Y 163 A E G K L Q R S T 164 L M V W 165 A C D F N R S V V WW Y 166 A C E F G H L N Q 167 A D G H K M N P 168 H R T W Y R S T Y 169L R V 170 A Q N R V 171 I V 172 A C 173 Q N R 174 A G H K M N Q RS T V W Y 175 E H T V Y 176 K L 177 V 178 G K M R 179 A C E G I K L M N180 F G I K M 181 K M Q P R S T V 182 L 183 E L 184 W 186 Y 192 S T 193F G Q R S Y 195 A G H I L N Q R S T W V 196 E G L N R S T W Y 197A D E F G H K L M 198 A D E H L N Q R S Q R S T W T W Y 200 D T 202 M204 P W 205 L R S T V W Y 206 H I K L M Q R S T 208 A C K L M Q R S T V209 A E F G L N R S T 211 L W 212 N S T 213 A E G H K L M N Q R V W Y 214 Q 215 A D E G H K L M 217 M Q R T V W Y  218 F M V 219A C D E H I K L M 220 A D H I L M S T V R S T W 221 A C I M Q T V 222D F G I K L N R S 224 I V 226 W 230 I 231 T 232 S 233 A F G K L R Y 234L M 235 A E G H K T 236 A G H K R S 237 A C E F H L N Q R 238D E H K Q R S T S T W 239 N 240 K A M P Q R S V 242 F 245 H 247 I L M248 A H W Y 251 L M Y 253 I 255 A G N Q R S 256 A H L V 257A C G I K L M N Q 258 G H N R S 259 E G I K L N P Q R R T V S T V W Y260 A D E G H L M Q R 261 A F K M N Q R T V 263 A H K M R T V S Y W 264A H 265 I 266 Y 267 M T 269 A C D S 270 M N S T 271 F G L M S V 272D M R S T 273 H T Y 274 A F S 275 L V 276 C D E G H I L M R 277A C D E G H K M S Y N Q R S T Y 278 A E F G H I K N R 279 A H Q R T 280GQ S T V Y 282 D G M Q 283 E P R S T 284 A E G H L M N Q S T Y 285AFGHMNQY 286 RS W 287 I N T 288 289 KS 290 I M 291 C Q R S V 292A C F G H K N P R 293 A C D F G K L M V W P Q S V Y 294 M 297 A 298 G I300 R 301 A V 302 I W 303 D V 304 G I 305 D E N 306 D E S 307G K N Q S T V W Y 308 D G H K N P R T 309 D E G H K L M N Q R S T V W310 A F G Q R S V Y 311 G H K Q S T 312 G K L N T 313 A E G H K L P R S314 A D H I N Q R S T 315 A E G H K L M R T V Y Y T Y 316 D 317A D H I K M N Q R 318 D F G H I K M N Q S T W R S T 320E G H I K L M N R 321 A D H K R S T Y 323 F I L S W V Y 324A D H M N R S 325 A D E G H K M N 326 C K L V Y Q S V W 327 M 328A C G H I K L Q R 331 C E V S T V W Y 334 P T 335 S 338 Q 339 M 342 A343 T V 347 A E G L M R S 348 D G S 349 A E K M N R T 351 A C I Q S 353T V 356 A D H S 357 A C K S T 358 C G L T 359 D E H K M T V 360 T 361 H367 A C G K R S 368 A E G H K L M R S T V H R S 371 E F G H I K L M R373 A E F K L M R S V 374 A H I M N P R S T S V V W Y 375A G I K L M N R S 376 A D E L M Q R S T 377 D E H K P R S T T V Y 378K N R 379 G H R S T 380 I L P T V W Y 381 E H K N Q R S V 383A E H I K L M N S 385 A G H N Q R S T T V V 387 S 388 F H I M R T V W Y389 A G H K L M P Q R S T Y 391 C 392 A F G K L M Q R S 393A D F H K L M N T V W Y R S T 394 L W 395 A G H K R T W 396A D H L Q R S T 397 R 398 L 399 A C E K M N Q R S 401 A E G Q N 403 FT V W 404 A P T 405 A F G K M P Q R S 406 A C E F G I N Q S W Y T V Y407 A D E F G H L M N 409 A D E G H I P Q R 410 D K M N P Q R SP Q R V W S T V T V Y 411 A H N P R S T V 412 D G H I L N Q P R 413A E H K N Q R S S V W Y T 414 I K L M 415 G S W V Y 416F G H I K L N Q R T V Y 417 I 418 A E F G I L M N P 419E F G H I K L N R Q R S V Y S W Y 420 I P 421 A E G H I K L M N 422 I TQ R S T Y 425 G I K M N R S Y 426 E G K N P Q S Y 427 H I K Q S T 428L M P T 431 A E G H I K L N Q 432 E G H N S V R S V W Y 433A C D E G H I K L 434 F G I M V 435 A C E G H R S T V P R S T V W Y 436C D E G H I K L M 437 A D G H I K L M Q 438 A C D E G L N P QQ R S T W Y R S Y R S T V W 439 A C F G H K L P Q 440 A D E F G H I L M441 A D F G H K L N S T V W P R S V Y Q S T V Y 442 C G H K L P Q R T443 A E F G H L M N Q 444 D E F G H I K M N V W Y R S T W R V W Y 445A G H L M N P Q R 446 A C D E G H I K L 447 D E F G I L M N P S T V W YM Q R T V W Q R T V W

In particular examples, provided herein is a modified PH20 polypeptidecontaining an amino acid replacement or replacements at a position orpositions corresponding to 1, 6, 8, 9, 10, 11, 12, 14, 15, 20, 22, 24,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 46, 47,48, 49, 50, 52, 58, 59, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 79, 82,83, 84, 86, 87, 89, 90, 92, 93, 94, 97, 102, 104, 107, 114, 118, 120,127, 128, 130, 131, 132, 135, 138, 139, 140, 141, 142, 143, 144, 146,147, 148, 149, 150, 151, 152, 155, 156, 158, 160, 162, 163, 164, 165,166, 167, 169, 170, 172, 173, 174, 175, 178, 179, 193, 195, 196, 198,204, 205, 206, 209, 212, 213, 215, 219, 220, 221, 222, 232, 233, 234,235, 236, 237, 238, 240, 247, 248, 249, 257, 258, 259, 260, 261, 263,267, 269, 271, 272, 273, 274, 276, 277, 278, 279, 282, 283, 285, 287,289, 291, 292, 293, 298, 305, 307, 308, 309, 310, 313, 314, 315, 317,318, 320, 321, 324, 325, 326, 328, 335, 347, 349, 351, 353, 356, 359,367, 368, 369, 371, 373, 374, 375, 376, 377, 380, 381, 383, 385, 389,392, 393, 395, 396, 399, 401, 404, 405, 406, 407, 409, 410, 412, 416,418, 419, 421, 425, 427, 428, 431, 433, 436, 437, 438, 439, 440, 441,442, 443, 444, 445, 446 or 447 with reference to amino acid positionsset forth in SEQ ID NO:3. For example, the amino acid positions can bereplacements at positions corresponding to replacement of Leucine (L) atposition 1 (L1), P6, V8, I9, P10, N11, V12, F14, L15, A20, S22, F24,L26, G27, K28, F29, D30, E31, P32, L33, D34, M35, S36, L37, F38, S39,F40, I41, I46, N47, A48, T49, G50, G52, V58, D59, Y63, I67, D68, S69,I70, T71, G72, V73, T74, V75, I79, K82, I83, S84, G86, D87, L89, D90,A92, K93, K94, T97, V102, N104, M107, E114, T118, A120, D127, V128,K130, N131, R132, E135, Q138, Q139, Q140, N141, V142, Q143, L144, L146,T147, E148, A149, T150, E151, K152, Q155, E156, E158, A160, K162, D163,F164, L165, V166, E167, 1169, K170, G172, K173, L174, L175, N178, H179,H193, K195, K196, G198, F204, N205, V206, K209, D212, D213, S215, N219,E220, S221, T222, T232, Q233, Q234, S235, P236, V237, A238, T240, V247,R248, E249, P257, D258, A259, K260, S261, L263, A267, T269, 1271, V272,F273, T274, Q276, V277, L278, K279, S282, Q283, E285, V287, T289, G291,E292, T293, A298, G305, L307, S308, I309, M310, M313, K314, S315, L317,L318, D320, N321, E324, T325, I326, N328, T335, Q347, Q349, V351, I353,N356, S359, P367, D368, N369, A371, Q373, L374, E375, K376, G377, F380,T381, R383, K385, E389, E392, Q393, S395, E396, Y399, S401, S404, T405,L406, S407, K409, E410, A412, D416, D418, A419, D421, A425, G427, A428,D431, F433, P436, P437, M438, E439, T440, E441, E442, P443, Q444, I445,F446 or Y447 with reference to amino acid positions set forth in SEQ IDNO:3.

Exemplary amino acid replacements in the modified PH20 polypeptidesprovided herein include, but are not limited, replacement with:histidine (H) at a position corresponding to position 1; A at a positioncorresponding to position 1; E at a position corresponding to position1; G at a position corresponding to position 1; K at a positioncorresponding to position 1; Q at a position corresponding to position1; Rat a position corresponding to position 1; A at a positioncorresponding to position 6; M at a position corresponding to position8; Q at a position corresponding to position 9; G at a positioncorresponding to position 10; H at a position corresponding to position10; S at a position corresponding to position 11; E at a positioncorresponding to position 12; I at a position corresponding to position12; K at a position corresponding to position 12; T at a positioncorresponding to position 12; V at a position corresponding to position14; V at a position corresponding to position 15; M at a positioncorresponding to position 15; S at a position corresponding to position20; T at a position corresponding to position 22; E at a positioncorresponding to position 24; H at a position corresponding to position24; R at a position corresponding to position 24; A at a positioncorresponding to position 26; E at a position corresponding to position26; K at a position corresponding to position 26; M at a positioncorresponding to position 26; Q at a position corresponding to position26; R at a position corresponding to position 26; D at a positioncorresponding to position 27; K at a position corresponding to position27; R at a position corresponding to position 27; R at a positioncorresponding to position 28; E at a position corresponding to position29; I at a position corresponding to position 29; K at a positioncorresponding to position 29; L at a position corresponding to position29; M at a position corresponding to position 29; P at a positioncorresponding to position 29; R at a position corresponding to position29; S at a position corresponding to position 29; T at a positioncorresponding to position 29; V at a position corresponding to position29; G at a position corresponding to position 30; H at a positioncorresponding to position 30; K at a position corresponding to position30; L at a position corresponding to position 30; M at a positioncorresponding to position 30; Rat a position corresponding to position30; S at a position corresponding to position 30; A at a positioncorresponding to position 31; C at a position corresponding to position31; G at a position corresponding to position 31; H at a positioncorresponding to position 31; I at a position corresponding to position31; K at a position corresponding to position 31; L at a positioncorresponding to position 31; P at a position corresponding to position31; R at a position corresponding to position 31; S at a positioncorresponding to position 31; T at a position corresponding to position31; V at a position corresponding to position 31; W at a positioncorresponding to position 31; C at a position corresponding to position32; F at a position corresponding to position 32; G at a positioncorresponding to position 32; H at a position corresponding to position32; W at a position corresponding to position 33; G at a positioncorresponding to position 33; W at a position corresponding to position34; Q at a position corresponding to position 35; V at a positioncorresponding to position 35; H at a position corresponding to position36; N at a position corresponding to position 36; F at a positioncorresponding to position 37; M at a position corresponding to position37; Y at a position corresponding to position 38; A at a positioncorresponding to position 39; L at a position corresponding to position39; N at a position corresponding to position 39; T at a positioncorresponding to position 39; L at a position corresponding to position40; T at a position corresponding to position 41; L at a positioncorresponding to position 46; Rat a position corresponding to position46; D at a position corresponding to position 47; F at a positioncorresponding to position 47; T at a position corresponding to position47; W at a position corresponding to position 47, with F at a positioncorresponding to position 48; H at a position corresponding to position48; K at a position corresponding to position 48; N at a positioncorresponding to position 48; R at a position corresponding to position49; D at a position corresponding to position 50; S at a positioncorresponding to position 50; M at a position corresponding to position50; N at a position corresponding to position 52; Q at a positioncorresponding to position 52; R at a position corresponding to position52; S at a position corresponding to position 52; T at a positioncorresponding to position 52; C at a position corresponding to position58; K at a position corresponding to position 58; L at a positioncorresponding to position 58; P at a position corresponding to position58; Q at a position corresponding to position 58; Rat a positioncorresponding to position 58; H at a position corresponding to position58; N at a position corresponding to position 58; Y at a positioncorresponding to position 58; N at a position corresponding to position59; K at a position corresponding to position 63; L at a positioncorresponding to position 63; M at a position corresponding to position63; Rat a position corresponding to position 63; W at a positioncorresponding to position 63; V at a position corresponding to position67; H at a position corresponding to position 68; P at a positioncorresponding to position 68; Q at a position corresponding to position68; A at a position corresponding to position 69; C at a positioncorresponding to position 69; E at a position corresponding to position69; F at a position corresponding to position 69; G at a positioncorresponding to position 69; I at a position corresponding to position69; L at a position corresponding to position 69; M at a positioncorresponding to position 69; P at a position corresponding to position69; R at a position corresponding to position 69; T at a positioncorresponding to position 69; W at a position corresponding to position69; Y at a position corresponding to position 69; A at a positioncorresponding to position 70; C at a position corresponding to position70; F at a position corresponding to position 70; G at a positioncorresponding to position 70; H at a position corresponding to position70; K at a position corresponding to position 70; L at a positioncorresponding to position 70; N at a position corresponding to position70; P at a position corresponding to position 70; R at a positioncorresponding to position 70; S at a position corresponding to position70; T at a position corresponding to position 70; V at a positioncorresponding to position 70; Y at a position corresponding to position70; G at a position corresponding to position 71; N at a positioncorresponding to position 71; R at a position corresponding to position71; S at a position corresponding to position 71; K at a positioncorresponding to position 72; M at a position corresponding to position72; Q at a position corresponding to position 72; A at a positioncorresponding to position 73; H at a position corresponding to position73; K at a position corresponding to position 73; L at a positioncorresponding to position 73; Q at a position corresponding to position73; Rat a position corresponding to position 73; Tat a positioncorresponding to position 73; W at a position corresponding to position73; A at a position corresponding to position 74; C at a positioncorresponding to position 74; E at a position corresponding to position74; F at a position corresponding to position 74; G at a positioncorresponding to position 74; H at a position corresponding to position74; K at a position corresponding to position 74; L at a positioncorresponding to position 74; M at a position corresponding to position74; N at a position corresponding to position 74; P at a positioncorresponding to position 74; R at a position corresponding to position74; S at a position corresponding to position 74; V at a positioncorresponding to position 74; W at a position corresponding to position74; F at a position corresponding to position 75; L at a positioncorresponding to position 75; M at position corresponding to position75; Rat a position corresponding to position 75; T at a positioncorresponding to position 75; L at a position corresponding to position79; L at a position corresponding to position 82; N at a positioncorresponding to position 82; V at a position corresponding to position83; Q at a position corresponding to position 83; S at a positioncorresponding to position 83; G at a position corresponding to position83; E at a position corresponding to position 84; F at a positioncorresponding to position 84; G at a position corresponding to position84; N at a position corresponding to position 84; R at a positioncorresponding to position 84; A at a position corresponding to position86; H at a position corresponding to position 86; K at a positioncorresponding to position 86; N at a position corresponding to position86; S at a position corresponding to position 86; T at a positioncorresponding to position 86; W at a position corresponding to position86; C at a position corresponding to position 87; G at a positioncorresponding to position 87; L at a position corresponding to position87; M at a position corresponding to position 87; R at a positioncorresponding to position 87; S at a position corresponding to position87; T at a position corresponding to position 87; V at a positioncorresponding to position 87; Y at a position corresponding to position87; C at a position corresponding to position 89; A at a positioncorresponding to position 90; E at a position corresponding to position90; H at a position corresponding to position 90; K at a positioncorresponding to position 90; N at a position corresponding to position90; R at a position corresponding to position 90; C at a positioncorresponding to position 92; L at a position corresponding to position92; I at a position corresponding to position 93; L at a positioncorresponding to position 93; Q at a position corresponding to position93; Rat a position corresponding to position 93; S at a positioncorresponding to position 93; T at a position corresponding to position93; D at a position corresponding to position 94; Q at a positioncorresponding to position 94; R at a position corresponding to position94; A at a position corresponding to position 97; C at an amino acidresidue corresponding to position 97; D at a position corresponding toposition 97; E at a position corresponding to position 97; G at aposition corresponding to position 97; L at a position corresponding toposition 97; S at a position corresponding to position 97; S at aposition corresponding to position 102; T at a position corresponding toposition 102; Rat a position corresponding to position 104; L at aposition corresponding to position 107; A at a position corresponding toposition 114; Q at a position corresponding to position 118; H at aposition corresponding to position 120; F at a position corresponding toposition 120; I at a position corresponding to position 120; S at aposition corresponding to position 120; V at a position corresponding toposition 120; Y at a position corresponding to position 120; E at aposition corresponding to position 127; H at a position corresponding toposition 127; N at a position corresponding to position 127; Q at aposition corresponding to position 127; R at a position corresponding toposition 127; I at a position corresponding to position 128; R at aposition corresponding to position 130; G at a position corresponding toposition 131; I at a position corresponding to position 131; M at aposition corresponding to position 131; Q at a position corresponding toposition 131; Rat a position corresponding to position 131; V at aposition corresponding to position 131; N at a position corresponding toposition 132; L at a position corresponding to position 132; D at aposition corresponding to position 135; G at a position corresponding toposition 135; Rat a position corresponding to position 135, with L at aposition corresponding to position 138; T at a position corresponding toposition 139; K at a position corresponding to position 140; H at aposition corresponding to position 141; Rat a position corresponding toposition 141; S at a position corresponding to position 141; W at aposition corresponding to position 141; Y at a position corresponding toposition 141; D at a position corresponding to position 142; G at aposition corresponding to position 142; K at a position corresponding toposition 142; N at a position corresponding to position 142; P at aposition corresponding to position 142; Q at a position corresponding toposition 142; R at a position corresponding to position 142; S at aposition corresponding to position 142; T at a position corresponding toposition 142; G at a position corresponding to position 143; K at aposition corresponding to position 143; Rat a position corresponding toposition 144; T at a position corresponding to position 144; P at aposition corresponding to position 146; R at a position corresponding toposition 146; A at a position corresponding to position 147; F at aposition corresponding to position 147; L at a position corresponding toposition 147; R at a position corresponding to position 147; S at aposition corresponding to position 147; V at a position corresponding toposition 147; H at a position corresponding to position 148; K at aposition corresponding to position 148; Q at a position corresponding toposition 148; T at a position corresponding to position 149; V at aposition corresponding to position 149; A at a position corresponding toposition 150; D at a position corresponding to position 150; G at aposition corresponding to position 150; N at a position corresponding toposition 150; S at a position corresponding to position 150; W at aposition corresponding to position 150; Y at a position corresponding toposition 150; A at a position corresponding to position 151; H at aposition corresponding to position 151; K at a position corresponding toposition 151; L at a position corresponding to position 151; M at aposition corresponding to position 151; Q at a position corresponding toposition 151; R at a position corresponding to position 151; S at aposition corresponding to position 151; T at a position corresponding toposition 151; V at a position corresponding to position 151; W at aposition corresponding to position 151; Y at a position corresponding toposition 151; Rat a position corresponding to position 152; Tat aposition corresponding to position 152; W at a position corresponding toposition 152; D at a position corresponding to position 155; G at aposition corresponding to position 155; K at a position corresponding toposition 155; Rat a position corresponding to position 155; D at aposition corresponding to position 156; Q at a position corresponding toposition 158; S at a position corresponding to position 158; S at aposition corresponding to position 160; E at a position corresponding toposition 162; A at a position corresponding to position 163; E at aposition corresponding to position 163; K at a position corresponding toposition 163; Q at a position corresponding to position 163; Rat aposition corresponding to position 163; S at a position corresponding toposition 163; M at a position corresponding to position 164; V at aposition corresponding to position 164; D at a position corresponding toposition 165; F at a position corresponding to position 165; N at aposition corresponding to position 165; S at a position corresponding toposition 165; V at a position corresponding to position 165; A at aposition corresponding to position 166; E at a position corresponding toposition 166; F at a position corresponding to position 166; H at aposition corresponding to position 166; L at a position corresponding toposition 166; Q at a position corresponding to position 166; Rat aposition corresponding to position 166; T at a position corresponding toposition 166; W at a position corresponding to position 166; Y at aposition corresponding to position 166; D at a position corresponding toposition 167; L at a position corresponding to position 169; Rat aposition corresponding to position 170; A at a position corresponding toposition 172; Rat a position corresponding to position 173; G at aposition corresponding to position 174; K at a position corresponding toposition 174; N at a position corresponding to position 174; Rat aposition corresponding to position 174; T at a position corresponding toposition 174; T at a position corresponding to position 175; K at aposition corresponding to position 178; Rat a position corresponding toposition 178; K at a position corresponding to position 179; Q at aposition corresponding to position 193; T at a position corresponding toposition 195; N at a position corresponding to position 195; with E at aposition corresponding to position 196; R at a position corresponding toposition 196; with D at a position corresponding to position 198; P at aposition corresponding to position 204; A at a position corresponding toposition 205; E at a position corresponding to position 205; L at aposition corresponding to position 205; T at a position corresponding toposition 205; I at a position corresponding to position 206; K at aposition corresponding to position 206; L at a position corresponding toposition 206; R at a position corresponding to position 206; R at aposition corresponding to position 209; N at a position corresponding toposition 212; S at a position corresponding to position 212; A at aposition corresponding to position 213; M at a position corresponding toposition 213; N at a position corresponding to position 213; H at aposition corresponding to position 215; M at a position corresponding toposition 215; A at a position corresponding to position 219; I at aposition corresponding to position 219; K at a position corresponding toposition 219; S at a position corresponding to position 219; H at aposition corresponding to position 220; I at a position corresponding toposition 220; L at a position corresponding to position 220; V at aposition corresponding to position 220; Q at a position corresponding toposition 221; G at a position corresponding to position 222; F at aposition corresponding to position 232; G at a position corresponding toposition 233; K at a position corresponding to position 233; Rat aposition corresponding to position 233; M at a position corresponding toposition 234; A at a position corresponding to position 235; R at aposition corresponding to position 236; C at a position corresponding toposition 237; E at a position corresponding to position 237; H at aposition corresponding to position 237; Q at a position corresponding toposition 237; T at a position corresponding to position 237; E at aposition corresponding to position 238; H at a position corresponding toamino acid position 238; S at a position corresponding to position 238;A at a position corresponding to position 240; Q at a positioncorresponding to position 240; I at a position corresponding to position247; A at a position corresponding to position 248; V at a positioncorresponding to position 249; G at a position corresponding to position257; Tat a position corresponding to position 257; Rat a positioncorresponding to position 257; N at a position corresponding to position258; S at a position corresponding to position 258; P at a positioncorresponding to position 259; M at a position corresponding to position260; Y at a position corresponding to position 260; A at a positioncorresponding to position 261; K at a position corresponding to position261; N at a position corresponding to position 261; K at a positioncorresponding to position 263; Rat a position corresponding to position263; T at a position corresponding to position 267; A at a positioncorresponding to position 269; L at a position corresponding to position271; M at a position corresponding to position 271; D at a positioncorresponding to position 272; Tat a position corresponding to position272; H at a position corresponding to position 273; Y at a positioncorresponding to position 273; F at a position corresponding to position274; D at a position corresponding to position 276; H at a positioncorresponding to position 276; M at a position corresponding to position276; R at a position corresponding to position 276; S at a positioncorresponding to position 276; Y at a position corresponding to position276; A at a position corresponding to position 277; E at a positioncorresponding to position 277; H at a position corresponding to position277; K at a position corresponding to position 277; M at a positioncorresponding to position 277; N at a position corresponding to position277; Q at a position corresponding to position 277; R at a positioncorresponding to position 277; S at a position corresponding to position277; T at a position corresponding to position 277; E at a positioncorresponding to position 278; F at a position corresponding to position278; G at a position corresponding to position 278; H at a positioncorresponding to position 278; K at a position corresponding to position278; N at a position corresponding to position 278; R at a positioncorresponding to position 278; S at a position corresponding to position278; T at a position corresponding to position 278; Y at a positioncorresponding to position 278; H at a position corresponding to position279; M at a position corresponding to position 282; S at a positioncorresponding to position 283; H at a position corresponding to position285; T at a position corresponding to position 287; S at a positioncorresponding to position 289; S at a position corresponding to position291; V at a position corresponding to position 291; C at a positioncorresponding to position 292; F at a position corresponding to position292; H at a position corresponding to position 292; K at a positioncorresponding to position 292; R at a position corresponding to position292; V at a position corresponding to position 292; A at a positioncorresponding to position 293; C at a position corresponding to position293; D at a position corresponding to position 293; F at a positioncorresponding to position 293; K at a position corresponding to position293; M at a position corresponding to position 293; P at a positioncorresponding to position 293; Q at a position corresponding to position293; V at a position corresponding to position 293; Y at a positioncorresponding to position 293; G at a position corresponding to position298; E at a position corresponding to position 305; G at a positioncorresponding to position 307; D at a position corresponding to position308; G at a position corresponding to position 308; K at a positioncorresponding to position 308; N at a position corresponding to position308; Rat a position corresponding to position 308; E at a positioncorresponding to position 309; G at a position corresponding to position309; H at a position corresponding to position 309; L at a positioncorresponding to position 309; M at a position corresponding to position309; N at a position corresponding to position 309; Q at a positioncorresponding to position 309; Rat a position corresponding to position309; S at a position corresponding to position 309; T at a positioncorresponding to position 309; V at a position corresponding to position309; A at a position corresponding to position 310; G at a positioncorresponding to position 310; Q at a position corresponding to position310; S at a position corresponding to position 310; A at a positioncorresponding to position 313; G at a position corresponding to position313; H at a position corresponding to position 313; K at a positioncorresponding to position 313; P at a position corresponding to position313; Rat a position corresponding to position 313; Tat a positioncorresponding to position 313; Y at a position corresponding to position313; with S at a position corresponding to position 314; Y at a positioncorresponding to position 314; A at a position corresponding to position315; H at a position corresponding to position 315; Y at a positioncorresponding to position 315; A at a position corresponding to position317; I at a position corresponding to position 317; K at a positioncorresponding to position 317; N at a position corresponding to position317; Q at a position corresponding to position 317; Rat a positioncorresponding to position 317; S at a position corresponding to position317; T at a position corresponding to position 317; W at a positioncorresponding to position 317; D at a position corresponding to position318; H at a position corresponding to position 318; K at a positioncorresponding to position 318; M at a position corresponding to position318; R at a position corresponding to position 318; H at a positioncorresponding to position 320; K at a position corresponding to position320; R at a position corresponding to position 320; Rat a positioncorresponding to position 321; S at a position corresponding to position321; N at a position corresponding to position 324; Rat a positioncorresponding to position 324; A at a position corresponding to position325; D at a position corresponding to position 325; E at a positioncorresponding to position 325; G at a position corresponding to position325; H at a position corresponding to position 325; K at a positioncorresponding to position 325; M at a position corresponding to position325; N at a position corresponding to position 325; Q at a positioncorresponding to position 325; S at a position corresponding to position325; V at a position corresponding to position 325; L at a positioncorresponding to position 326; V at a position corresponding to position326; C at a position corresponding to position 328; G at a positioncorresponding to position 328; I at a position corresponding to position328; K at a position corresponding to position 328; L at a positioncorresponding to position 328; S at a position corresponding to position328; Y at a position corresponding to position 328; S at a positioncorresponding to position 335; A at a position corresponding to position347; G at a position corresponding to position 347; S at a positioncorresponding to position 347; M at a position corresponding to position349; R at a position corresponding to position 349; S at a positioncorresponding to position 351; V at a position corresponding to position353; with H at a position corresponding to position 356; S at a positioncorresponding to position 356; E at a position corresponding to position359; H at a position corresponding to position 359; T at a positioncorresponding to position 359; A at a position corresponding to position367; G at a position corresponding to position 367; K at a positioncorresponding to position 367; S at a position corresponding to position367; A at a position corresponding to position 368; E at a positioncorresponding to position 368; K at a position corresponding to position368; L at a position corresponding to amino acid position 368; M at aposition corresponding to amino acid position 368; R at a positioncorresponding to position 368; Tat a position corresponding to aminoacid position 368; H at a position corresponding to position 369; R at aposition corresponding to position 369; F at a position corresponding toposition 371; H at a position corresponding to position 371; K at aposition corresponding to position 371; L at a position corresponding toposition 371; Rat a position corresponding to position 371; S at aposition corresponding to position 371; M at a position corresponding toposition 373; H at a position corresponding to position 374; P at aposition corresponding to position 374; A at a position corresponding toposition 375; G at a position corresponding to position 375; K at aposition corresponding to position 375; Rat a position corresponding toposition 375; D at a position corresponding to position 376; E at aposition corresponding to position 376; Q at a position corresponding toposition 376; R at a position corresponding to position 376; T at aposition corresponding to position 376; V at a position corresponding toposition 376; Y at a position corresponding to position 376; D at aposition corresponding to position 377; E at a position corresponding toposition 377; H at a position corresponding to position 377; K at aposition corresponding to position 377; P at a position corresponding toposition 377; R at a position corresponding to position 377; S at aposition corresponding to position 377; T at a position corresponding toposition 377; W at a position corresponding to position 380; Y at aposition corresponding to position 380; S at a position corresponding toposition 381; I at a position corresponding to position 383; K at aposition corresponding to position 383; L at a position corresponding toposition 383; S at a position corresponding to position 383; A at aposition corresponding to position 385; Q at a position corresponding toposition 385; V at a position corresponding to position 385; A at aposition corresponding to position 389; G at a position corresponding toposition 389; L at a position corresponding to position 389; K at aposition corresponding to position 389; Q at a position corresponding toposition 389; S at a position corresponding to position 389; A at aposition corresponding to position 392; F at a position corresponding toposition 392; M at a position corresponding to position 392; Q at aposition corresponding to position 392; Rat a position corresponding toposition 392; V at a position corresponding to position 392; F at aposition corresponding to position 393; M at a position corresponding toposition 393; A at a position corresponding to position 395; H at aposition corresponding to position 395; Rat a position corresponding toposition 395; A at a position corresponding to position 396; H at aposition corresponding to position 396; Q at a position corresponding toposition 396; S at a position corresponding to position 396; K at aposition corresponding to position 399; M at a position corresponding toposition 399; T at a position corresponding to position 399; V at aposition corresponding to position 399; W at a position corresponding toposition 399; A at a position corresponding to position 401; E at aposition corresponding to position 401; A at a position corresponding toposition 404; G at a position corresponding to position 405; F at aposition corresponding to position 406; N at a position corresponding toposition 406; A at a position corresponding to position 407; D at aposition corresponding to position 407; E at a position corresponding toposition 407; F at a position corresponding to position 407; H at aposition corresponding to position 407; Q at a position corresponding toposition 407; P at a position corresponding to position 407; A at aposition corresponding to position 409; Q at a position corresponding toposition 409; Tat a position corresponding to position 410; Q at aposition corresponding to position 412; Rat a position corresponding toposition 412; V at a position corresponding to position 412; L at aposition corresponding to position 416; E at a position corresponding toposition 418; L at a position corresponding to position 418; P at aposition corresponding to position 418; Rat a position corresponding toposition 418; V at a position corresponding to position 418; F at aposition corresponding to position 419; H at a position corresponding toposition 419; I at a position corresponding to position 419; K at aposition corresponding to position 419; Rat a position corresponding toposition 419; S at a position corresponding to position 419; Y at aposition corresponding to position 419; A at a position corresponding toposition 421; H at a position corresponding to position 421; K at aposition corresponding to position 421; N at a position corresponding toposition 421; Q at a position corresponding to position 421; Rat aposition corresponding to position 421; S at a position corresponding toposition 421; G at a position corresponding to position 425; K at aposition corresponding to position 425; Q at a position corresponding toposition 427; T at a position corresponding to position 427; L at aposition corresponding to position 428; A at a position corresponding toposition 431; G at a position corresponding to position 431; E at aposition corresponding to position 431; H at a position corresponding toposition 431; K at a position corresponding to position 431; L at aposition corresponding to position 431; N at a position corresponding toposition 431; Q at a position corresponding to position 431; Rat aposition corresponding to position 431; S at a position corresponding toposition 431; V at a position corresponding to position 431; A at aposition corresponding to position 433; H at a position corresponding toposition 433; I at a position corresponding to position 433; K at aposition corresponding to position 433; L at a position corresponding toposition 433; Rat a position corresponding to position 433; T at aposition corresponding to position 433; V at a position corresponding toposition 433; W at a position corresponding to position 433; K at aposition corresponding to position 436; I at a position corresponding toposition 437; M at a position corresponding to position 437; A at aposition corresponding to position 438; D at a position corresponding toposition 438; E at a position corresponding to position 438; L at aposition corresponding to position 438; N at a position corresponding toposition 438; T at a position corresponding to position 438; A at aposition corresponding to position 439; C at a position corresponding toposition 439; K at a position corresponding to position 439; P at aposition corresponding to position 439; Q at a position corresponding toposition 439; T at a position corresponding to position 439; V at aposition corresponding to position 439; D at a position corresponding toposition 440; H at a position corresponding to position 440; M at aposition corresponding to position 440; P at a position corresponding toposition 440; R at a position corresponding to position 440; S at aposition corresponding to position 440; A at a position corresponding toposition 441; F at a position corresponding to position 441; C at aposition corresponding to position 442; G at a position corresponding toposition 442; R at a position corresponding to position 442; A at aposition corresponding to position 443; E at a position corresponding toposition 443; F at a position corresponding to position 443; G at aposition corresponding to position 443; M at a position corresponding toposition 443; N at a position corresponding to position 443; E at aposition corresponding to position 444; H at a position corresponding toposition 444; V at a position corresponding to position 444; H at aposition corresponding to position 445; M at a position corresponding toposition 445; N at a position corresponding to position 445; P at aposition corresponding to position 445; Q at a position corresponding toposition 445; S at a position corresponding to position 445; T at aposition corresponding to position 445; V at a position corresponding toposition 445; W at a position corresponding to position 445; A at aposition corresponding to position 446; M at a position corresponding toposition 446; W at a position corresponding to position 446; D at aposition corresponding to position 447; E at a position corresponding toposition 447; G at a position corresponding to position 447; I at aposition corresponding to position 447; N at a position corresponding toposition 447; P at a position corresponding to position 447; Q at aposition corresponding to position 447; T at a position corresponding toposition 447, and/or replacement with V at a position corresponding toposition 447, each with reference to amino acid positions set forth inSEQ ID NO:3.

Exemplary of such modified PH20 polypeptides are any having the sequenceof amino acids set forth in any of SEQ ID NOs: 74-855, or having asequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to any of SEQ ID NOs:74-855 and contains theamino acid replacement and exhibits hyaluronidase activity.

Any of the above modified PH20 polypeptides provided herein can exhibitaltered, such as improved or increased, properties or activitiescompared to the corresponding PH20 polypeptide not containing the aminoacid modification (e.g., amino acid replacement). For example, thealtered activities or properties can be an increased catalytic activityand/or an increased stability under denaturing conditions.

a. In Creased Activity

Provided herein are modified or variant PH20 polypeptides that containone or more amino acid replacements in a PH20 polypeptide and thatexhibit increased hyaluronidase activity compared to the correspondingPH20 polypeptide not containing the amino acid replacement(s), forexample, the PH20 polypeptide set forth in any of SEQ ID NOs: 2, 3,6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. Inparticular, the modified or variant PH20 polypeptides provided hereinexhibit increased hyaluronidase activity compared to the correspondingPH20 polypeptide not containing the amino acid replacement, for example,the PH20 polypeptide set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or72 and in particular the PH20 polypeptide set forth in SEQ ID NO:3.

The modified PH20 polypeptide can exhibit hyaluronidase activity that isat least or about at least or 120%, 130%, 135%, 140%, 145%, 150%, 160%,170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%,4000%, 5000% of the hyaluronidase activity of the corresponding PH20polypeptide not containing the amino acid replacement(s), for examplethe PH20 polypeptide set forth in any of any of SEQ ID NOs: 2, 3, 6-66,68-72, 856-861, 869 or 870 or a variant thereof, under the sameconditions. For example, the hyaluronidase activity is increased atleast or about at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold,25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 200-fold, 300-fold, 400-fold or more.

In particular examples, the modified PH20 polypeptides contain an aminoacid replacement at one or more amino acid positions identified as beingassociated with increased hyaluronidase activity. As described herein,such positions have been identified using mutagenesis and selection orscreening methods to identify those positions that result in increasedhyaluronidase activity. The PH20 polypeptide also can contain othermodifications, such as other amino acid replacements, that alone are notassociated with increased activity so long as the resulting modifiedPH20 polypeptide exhibits increased hyaluronidase activity compared tothe PH20 not containing the amino acid modification(s), such as aminoacid replacement(s). The modified PH20 polypeptide provided herein cancontain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,or more amino acid replacements. Additional modifications, such asinsertions or deletions, also can be included. The amino acidreplacement can be in a PH20 polypeptide as set forth in any of SEQ IDNOs: 2,3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof havingat least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitythereto. For example, the replacement(s) can be in a human PH20polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7,32-66, 69 or 72 or a variant thereof.

For example, the modified PH20 polypeptides provided herein contain anamino acid replacement (substitution) at one or more amino acidpositions corresponding to positions 1, 12, 15, 24, 26, 27, 29, 30, 31,32, 33, 37, 39, 46, 48, 52, 58, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75,84, 86, 87, 92, 93, 94, 97, 118, 120, 127, 131, 135, 141, 142, 147, 148,150, 151, 152, 155, 156, 163, 164, 165, 166, 169, 170, 174, 198, 206,209, 212, 213, 215, 219, 233, 234, 236, 238, 247, 257, 259, 260, 261,263, 269, 271, 272, 276, 277, 278, 282, 291, 293, 305, 308, 309, 310,313, 315, 317, 318, 320, 324, 325, 326, 328, 347, 353, 359, 371, 377,380, 389, 392, 395, 399, 405, 407, 409, 410, 418, 419, 421, 425, 431,433, 436, 437, 438, 439, 440, 441, 442, 443, 445, 446 or 447 withreference to amino acid positions set forth in SEQ ID NO:3. For example,the amino acid positions can be replacements at positions correspondingto replacement of Leucine (L) at position 1 (L1), V12, L15, F24, L26,G27, F29, D30, E31, P32, L33, L37, S39, I46, A48, G52, V58, Y63, I67,D68, S69, I70, T71, G72, V73, T74, V75, S84, G86, D87, A92, K93, K94,T97, T118, A120, D127, N131, E135, N141, V142, T147, E148, T150, E151,K152, Q155, E156, D163, F164, L165, V166, 1169, K170, L174, G198, V206,K209, D212, D213, S215, N219, Q233, Q234, P236, A238, V247, P257, A259,K260, S261, L263, T269, 1271, V272, Q276, V277, L278, S282, G291, T293,G305, S308, 1309, M310, M313, S315, L317, L318, D320, E324, T325, 1326,N328, Q347, 1353, S359, A371, G377, F380, E389, E392, S395, Y399, T405,S407, K409, E410, D418, A419, D421, A425, D431, F433, P436, P437, M438,E439, T440, E441, E442, P443, 1445, F446 or Y447 with reference to aminoacid positions set forth in SEQ ID NO:3. Exemplary of such modified PH20polypeptides are polypeptides that exhibit at least 1.5-fold or more theactivity of the corresponding PH20 polypeptide not containing the aminoacid replacement.

Exemplary of amino acid replacements in the modified PH20 polypeptidesprovided herein include, but are not limited, replacement: withhistidine (H) at a position corresponding to position 1; Q at a positioncorresponding to position 1; E at a position corresponding to position12; T at a position corresponding to position 12; V at a positioncorresponding to position 15; E at a position corresponding to position24; H at a position corresponding to position 24; E at a positioncorresponding to position 26; K at a position corresponding to position26; K at a position corresponding to position 27; R at a positioncorresponding to position 27; E at a position corresponding to position29; I at a position corresponding to position 29; L at a positioncorresponding to position 29; M at a position corresponding to position29; P at a position corresponding to position 29; S at a positioncorresponding to position 29; V at a position corresponding to position29; G at a position corresponding to position 30; H at a positioncorresponding to position 30; K at a position corresponding to position30; M at a position corresponding to position 30; Rat a positioncorresponding to position 30; S at a position corresponding to position30; A at a position corresponding to position 31; C at a positioncorresponding to position 31; H at a position corresponding to position31; I at a position corresponding to position 31; K at a positioncorresponding to position 31; L at a position corresponding to position31; P at a position corresponding to position 31; R at a positioncorresponding to position 31; S at a position corresponding to position31; T at a position corresponding to position 31; V at a positioncorresponding to position 31; F at a position corresponding to position32; G at a position corresponding to position 32; H at a positioncorresponding to position 32; W at a position corresponding to position33; F at a position corresponding to position 37; N at a positioncorresponding to position 39; T at a position corresponding to position39; R at a position corresponding to position 46; F at a positioncorresponding to position 48; H at a position corresponding to position48; N at a position corresponding to position 48; Q at a positioncorresponding to position 52; K at a position corresponding to position58; Q at a position corresponding to position 58; W at a positioncorresponding to position 63; V at a position corresponding to position67; H at a position corresponding to position 68; Q at a positioncorresponding to position 68; A at a position corresponding to position69; C at a position corresponding to position 69; F at a positioncorresponding to position 69; G at a position corresponding to position69; I at a position corresponding to position 69; L at a positioncorresponding to position 69; M at a position corresponding to position69; P at a position corresponding to position 69; R at a positioncorresponding to position 69; W at a position corresponding to position69; Y at a position corresponding to position 69; A at a positioncorresponding to position 70; C at a position corresponding to position70; F at a position corresponding to position 70; G at a positioncorresponding to position 70; H at a position corresponding to position70; K at a position corresponding to position 70; L at a positioncorresponding to position 70; N at a position corresponding to position70; P at a position corresponding to position 70; R at a positioncorresponding to position 70; S at a position corresponding to position70; T at a position corresponding to position 70; V at a positioncorresponding to position 70; Rat a position corresponding to position71; S at a position corresponding to position 71; M at a positioncorresponding to position 72; Q at a position corresponding to position72; H at a position corresponding to position 73; L at a positioncorresponding to position 73; W at a position corresponding to position73; A at a position corresponding to position 74; C at a positioncorresponding to position 74; G at a position corresponding to position74; N at a position corresponding to position 74; P at a positioncorresponding to position 74; R at a position corresponding to position74; S at a position corresponding to position 74; V at a positioncorresponding to position 74; W at a position corresponding to position74; F at a position corresponding to position 75; L at a positioncorresponding to position 75; Rat a position corresponding to position75; T at a position corresponding to position 75; G at a positioncorresponding to position 84; Rat a position corresponding to position84; A at a position corresponding to position 86; C at a positioncorresponding to position 87; T at a position corresponding to position87; Y at a position corresponding to position 87; C at a positioncorresponding to position 92; I at a position corresponding to position93; L at a position corresponding to position 93; Rat a positioncorresponding to position 93; T at a position corresponding to position93; Rat a position corresponding to position 94; G at a positioncorresponding to position 97; Q at a position corresponding to position118; F at a position corresponding to position 120; V at a positioncorresponding to position 120; Y at a position corresponding to position120; H at a position corresponding to position 127; N at a positioncorresponding to position 127; G at a position corresponding to position131; R at a position corresponding to position 131; V at a positioncorresponding to position 131; D at a position corresponding to position135; G at a position corresponding to position 135; Rat a positioncorresponding to position 135, with H at a position corresponding toposition 141; Y at a position corresponding to position 141; Rat aposition corresponding to position 142; R at a position corresponding toposition 147; V at a position corresponding to position 147; K at aposition corresponding to position 148; G at a position corresponding toposition 150; K at a position corresponding to position 151; L at aposition corresponding to position 151; M at a position corresponding toposition 151; Q at a position corresponding to position 151; Rat aposition corresponding to position 151; Rat a position corresponding toposition 152; G at a position corresponding to position 155; K at aposition corresponding to position 155; D at a position corresponding toposition 156; A at a position corresponding to position 163; E at aposition corresponding to position 163; K at a position corresponding toposition 163; Rat a position corresponding to position 163; M at aposition corresponding to position 164; D at a position corresponding toposition 165; N at a position corresponding to position 165; A at aposition corresponding to position 166; F at a position corresponding toposition 166; H at a position corresponding to position 166; L at aposition corresponding to position 166; Q at a position corresponding toposition 166; Rat a position corresponding to position 166; Tat aposition corresponding to position 166; Y at a position corresponding toposition 166; L at a position corresponding to position 169; Rat aposition corresponding to position 170; K at a position corresponding toposition 174; D at a position corresponding to position 198; K at aposition corresponding to position 206; L at a position corresponding toposition 206; N at a position corresponding to position 212; M at aposition corresponding to position 213; N at a position corresponding toposition 213; M at a position corresponding to position 215; S at aposition corresponding to position 219; K at a position corresponding toposition 233; Rat a position corresponding to position 233; M at aposition corresponding to position 234; R at a position corresponding toposition 236; E at a position corresponding to position 237; S at aposition corresponding to position 238; I at a position corresponding toposition 247; T at a position corresponding to position 257; P at aposition corresponding to position 259; Y at a position corresponding toposition 260; K at a position corresponding to position 261; N at aposition corresponding to position 261; K at a position corresponding toposition 263; Rat a position corresponding to position 263; A at aposition corresponding to position 269; L at a position corresponding toposition 271; M at a position corresponding to position 271; Tat aposition corresponding to position 272; D at a position corresponding toposition 276; S at a position corresponding to position 276; Y at aposition corresponding to position 276; K at a position corresponding toposition 277; R at a position corresponding to position 277; T at aposition corresponding to position 277; H at a position corresponding toposition 278; K at a position corresponding to position 278; N at aposition corresponding to position 278; R at a position corresponding toposition 278; S at a position corresponding to position 278; T at aposition corresponding to position 278; Y at a position corresponding toposition 278; M at a position corresponding to position 282; V at aposition corresponding to position 291; A at a position corresponding toposition 293; C at a position corresponding to position 293; F at aposition corresponding to position 293; M at a position corresponding toposition 293; P at a position corresponding to position 293; Q at aposition corresponding to position 293; V at a position corresponding toposition 293; E at a position corresponding to position 305; G at aposition corresponding to position 308; N at a position corresponding toposition 308; E at a position corresponding to position 309; L at aposition corresponding to position 309; N at a position corresponding toposition 309; Q at a position corresponding to position 309; R at aposition corresponding to position 309; T at a position corresponding toposition 309; A at a position corresponding to position 310; G at aposition corresponding to position 310; K at a position corresponding toposition 313; Rat a position corresponding to position 313; H at aposition corresponding to position 315; I at a position corresponding toposition 317; K at a position corresponding to position 317; Rat aposition corresponding to position 317; M at a position corresponding toposition 318; H at a position corresponding to position 320; K at aposition corresponding to position 320; R at a position corresponding toposition 320; Rat a position corresponding to position 324; A at aposition corresponding to position 325; D at a position corresponding toposition 325; E at a position corresponding to position 325; G at aposition corresponding to position 325; H at a position corresponding toposition 325; K at a position corresponding to position 325; M at aposition corresponding to position 325; N at a position corresponding toposition 325; Q at a position corresponding to position 325; S at aposition corresponding to position 325; V at a position corresponding toposition 326; I at a position corresponding to position 328; K at aposition corresponding to position 328; L at a position corresponding toposition 328; S at a position corresponding to position 328; Y at aposition corresponding to position 328; G at a position corresponding toposition 347; S at a position corresponding to position 347; V at aposition corresponding to position 353; with T at a positioncorresponding to position 359; Rat a position corresponding to position371; P at a position corresponding to position 377; Tat a positioncorresponding to position 377; W at a position corresponding to position380; Y at a position corresponding to position 380; K at a positioncorresponding to position 389; M at a position corresponding to position392; Rat a position corresponding to position 395; M at a positioncorresponding to position 399; T at a position corresponding to position399; W at a position corresponding to position 399; G at a positioncorresponding to position 405; D at a position corresponding to position407; Q at a position corresponding to position 407; A at a positioncorresponding to position 409; Q at a position corresponding to position409; T at a position corresponding to position 410; P at a positioncorresponding to position 418; F at a position corresponding to position419; I at a position corresponding to position 419; K at a positioncorresponding to position 419; Rat a position corresponding to position419; S at a position corresponding to position 419; H at a positioncorresponding to position 421; K at a position corresponding to position421; N at a position corresponding to position 421; Q at a positioncorresponding to position 421; R at a position corresponding to position421; S at a position corresponding to position 421; K at a positioncorresponding to position 425; A at a position corresponding to position431; H at a position corresponding to position 431; K at a positioncorresponding to position 431; Q at a position corresponding to position431; R at a position corresponding to position 431; S at a positioncorresponding to position 431; V at a position corresponding to position431; L at a position corresponding to position 433; Rat a positioncorresponding to position 433; T at a position corresponding to position433; V at a position corresponding to position 433; K at a positioncorresponding to position 436; I at a position corresponding to position437; M at a position corresponding to position 437; T at a positioncorresponding to position 438; V at a position corresponding to position439; H at a position corresponding to position 440; R at a positioncorresponding to position 440; F at a position corresponding to position441; Rat a position corresponding to position 442; A at a positioncorresponding to position 443; M at a position corresponding to position443; M at a position corresponding to position 445; P at a positioncorresponding to position 445; A at a position corresponding to position446; D at a position corresponding to position 447; N at a positioncorresponding to position 447; and/or with Q at a position correspondingto position 447, each with reference to amino acid positions set forthin SEQ ID NO:3. The modified PH20 polypeptides can contain any one ormore of the recited amino acid substitutions, in any combination, withor without additional modifications, so long at the PH20 polypeptideexhibits hyaluronidase activity, such as increased hyaluronidaseactivity compared to the PH20 polypeptide not containing themodification(s), for example, at least 1.5-fold increased hyaluronidaseactivity.

In some examples, the modified PH20 polypeptides provided herein containone or more amino acid replacement(s) at a position(s) corresponding toposition(s) 24, 29, 31, 48, 58, 69, 70, 75, 84, 97, 165, 166, 271, 278,317, 320, 325, and/or 326 with reference to positions set forth in SEQID NO:3. For example, exemplary amino acid replacements include, but arenot limited to, replacement with: E at a position corresponding toposition 24; E at a position corresponding to position 29; V at aposition corresponding to position 31; N at a position corresponding toposition 48; K at a position corresponding to position 58; Q at aposition corresponding to position 58; A at a position corresponding toposition 69; F at a position corresponding to position 69; G at aposition corresponding to position 69; P at a position corresponding toposition 69; R at a position corresponding to position 69; A at aposition corresponding to position 70; F at a position corresponding toposition 70; G at a position corresponding to position 70; H at aposition corresponding to position 70; H at a position corresponding toposition 70; N at a position corresponding to position 70; R at aposition corresponding to position 70; T at a position corresponding toposition 70; V at a position corresponding to position 70; L at aposition corresponding to position 75; T at a position corresponding toposition 75; G at a position corresponding to position 84; G at aposition corresponding to position 97; D at a position corresponding toposition 165; L at a position corresponding to position 166; Rat aposition corresponding to position 166; T at a position corresponding toposition 166; L at a position corresponding to position 271; H at aposition corresponding to position 278; R at a position corresponding toposition 278; K at a position corresponding to position 317; K at aposition corresponding to position 320; E at a position corresponding toposition 325, with G at a position corresponding to position 325; K at aposition corresponding to position 325; N at a position corresponding toposition 325; Q at a position corresponding to position 325; V at aposition corresponding to position 326; each with reference to aminoacid positions set forth in SEQ ID NO:3. The modified PH20 polypeptidescan contain any one or more of the recited amino acid substitutions, inany combination, with or without additional modifications, so long atthe PH20 polypeptide exhibits hyaluronidase activity, such as increasedhyaluronidase activity compared PH20 polypeptide not containing themodification(s), for example, at least 2.0-fold increased hyaluronidaseactivity.

Exemplary modified PH20 polypeptides that exhibit increased activitycompared to the unmodified PH20 polypeptide (e.g., set forth in SEQ IDNO:3) are any having the sequence of amino acids set forth in any of SEQID NOs: 73, 78, 86, 89, 91, 95, 96, 99, 100, 105, 106, 108, 109, 111,112, 113, 115, 117, 118, 119, 120, 123-126, 128-136, 139-141, 149, 154,155, 159, 164, 165, 167, 173, 178, 181, 191-193, 195-197, 199-205,207-221, 225, 226, 228, 229, 231, 233, 237-239, 242, 247-254, 256, 257,267, 269, 270, 277, 283, 293, 295, 296, 298, 300, 303, 308, 316, 318,321, 322, 324, 325, 330, 334, 335, 338-340, 344, 348, 355, 367, 369,371, 377, 384-388, 394, 398, 399, 401, 406-408, 410, 412, 414, 416, 419,421-426, 428, 430, 431, 435, 448, 455, 456, 459, 462, 463, 465, 469,478-480, 482, 484, 490, 493, 497, 501, 503, 505, 506-508, 510-512, 514,518, 522, 523, 527, 531, 533, 537-543, 545, 551, 558, 559, 561, 563-566,569, 572, 574, 576, 579, 581-583, 585, 587, 588, 594, 596, 602, 605,606, 609, 613, 618-620, 624-634, 637, 640-644, 647, 648, 652, 657, 675,695, 698, 699, 700, 712, 717, 725, 731, 732, 734, 738, 742, 746,748-750, 757, 760, 762-765, 768-773, 775, 779, 782, 783, 786-789,794-797, 799-801, 807, 814, 816, 819, 822, 825, 826, 830, 836, 838, 844,847, 851, 853 or having a sequence of amino acids that exhibits at least68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:73, 78, 86, 89, 91, 95, 96, 99, 100, 105, 106, 108, 109, 111, 112, 113,115, 117, 118, 119, 120, 123-126, 128-136, 139-141, 149, 154, 155, 159,164, 165, 167, 173, 178, 181, 191-193, 195-197, 199-205, 207-221, 225,226, 228, 229, 231, 233, 237-239, 242, 247-254, 256, 257, 267, 269, 270,277, 283, 293, 295, 296, 298, 300, 303, 308, 316, 318, 321, 322, 324,325, 330, 334, 335, 338-340, 344, 348, 355, 367, 369, 371, 377, 384-388,394, 398, 399, 401, 406-408, 410, 412, 414, 416, 419, 421-426, 428, 430,431, 435, 448, 455, 456, 459, 462, 463, 465, 469, 478-480, 482, 484,490, 493, 497, 501, 503, 505, 506-508, 510-512, 514, 518, 522, 523, 527,531, 533, 537-543, 545, 551, 558, 559, 561, 563-566, 569, 572, 574, 576,579, 581-583, 585, 587, 588, 594, 596, 602, 605, 606, 609, 613, 618-620,624-634, 637, 640-644, 647, 648, 652, 657, 675, 695, 698, 699, 700, 712,717, 725, 731, 732, 734, 738, 742, 746, 748-750, 757, 760, 762-765,768-773, 775, 779, 782, 783, 786-789, 794-797, 799-801, 807, 814, 816,819, 822, 825, 826, 830, 836, 838, 844, 847, 851, 853 and contains theamino acid replacement and exhibits increased hyaluronidase activitycompared to the corresponding unmodified polypeptide.

b. Increased Stability

Provided herein are PH20 polypeptides that exhibit increased stability.In particular, the PH20 polypeptides exhibit increased stability in vivoand/or in vitro. For example, the PH20 polypeptides can exhibitincreased stability under various storage conditions. The modified PH20polypeptides provided herein that exhibit increased stability display,among other parameters, increased resistance to denaturation conditions,including but not limited to, denaturation conditions caused bytemperature (e.g., elevated temperature such as heat), agitation, no orlow salt, and/or presence of excipients. Exemplary excipients include,but are not limited to, antiadherents, binders, coatings, fillers anddiluents, flavors, colors, lubricants, glidants, preservatives, sorbentsor sweeteners. For example, various excipients, such as preservatives,can act as protein denaturing agents. Modified PH20 polypeptidesprovided herein that exhibit increased protein stability exhibit reducedaggregation, reduced precipitation and/or increased activity whenexposed to a denaturation condition compared to the corresponding PH20not containing the amino acid replacement. For example, modified PH20polypeptides provided herein exhibit at least or at least about or 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%,150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more increasedactivity when exposed to a denaturation condition compared to thecorresponding PH20 polypeptide not containing the amino acid replacementwhen exposed to the same denaturation condition.

The PH20 polypeptides provided herein that exhibit increased stabilityare modified or variant PH20 polypeptides that contain an amino acidreplacement (substitution), deletion or insertion or other modification.Typically, the PH20 polypeptides provided herein that exhibit increasedstability contain one or more amino acid replacements in a PH20polypeptide compared to the corresponding PH20 polypeptide notcontaining the amino acid replacement(s), for example, the PH20polypeptide set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861,869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity thereto. In particular, the modifiedor variant PH20 polypeptides provided herein exhibit increased stabilitycompared to the corresponding PH20 polypeptide not containing the aminoacid replacement, for example, the PH20 polypeptide set forth in any ofSEQ ID NOs: 3, 7, 32-66, 69 or 72 and in particular the PH20 polypeptideset forth in SEQ ID NO:3.

In particular examples, the modified PH20 polypeptides contain an aminoacid replacement at one or more amino acid positions identified as beingassociated with increased stability. As described herein, such positionscan be identified using mutagenesis and selection or screening methodsto identify those positions that result in stability (e.g., increasedactivity) of the polypeptide compared to the corresponding PH20 notcontaining the modification upon exposure to one or more denaturationconditions. The PH20 polypeptide also can contain other modifications,such as other amino acid replacements, that alone are not associatedwith conferring stability, so long as the resulting modified PH20polypeptide exhibits increased stability under one or more denaturationconditions compared to the PH20 not containing the amino acidmodification(s), such as amino acid replacement(s), and exhibitshyaluronidase activity. The modified PH20 polypeptide provided hereincan contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, or more amino acid replacements. Additional modifications, such asinsertions or deletions, also can be included. The amino acidreplacement can be in a PH20 polypeptide as set forth in any of SEQ IDNOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof havingat least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitythereto. For example, the replacements can be in a human PH20polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7,32-66, 69 or 72 or a variant thereof.

Exemplary of modified PH20 polypeptides provided herein are PH20polypeptides that exhibit increased stability upon exposure to phenolcompounds, high temperature (heat), and/or lack of NaCl.

i. Phenophiles

Provided herein are modified PH20 polypeptides that exhibit increasedstability in the presence of phenolic compounds. Multidose formulationsmust contain antimicrobial preservatives to protect them from microbialcontamination. For parenteral drug products, including insulin and othertherapeutic agents, the most common preservatives are phenoliccompounds, such as phenol, metacresol (m-cresol), benzyl alcohol, andparabens including methylparaben and propylparaben. The preservativestypically must be present at sufficient concentrations to satisfyregulatory rules. For example, regulatory requirements assert that theantimicrobial efficacy of the formulation must satisfy the preservativeefficacy test (PET) requirements of the target markets. Currentlydifferent regulatory agencies have different pharmacopeial criteria forantimicrobial effectiveness for pharmaceutical products designed formultiple dosing. The PET requirements of the United States Pharmacopoeia(USP) and the European Pharmacopoeia (EP) differ considerably, imposingadditional constraints in developing multidose formulations. Table 4shows the criteria for injectable drugs to meet USP and EP criteria.Typically, formulations that meet EP (EPA or EPB) anti-microbialrequirements contain more preservative than those formulated only tomeet USP anti-microbial requirements.

TABLE 4 USP and EP requirement for antimicrobial effectiveness testingUnited Europe Time States EPB EPA Requirement point USP (Minimum)(Preferred) Bacterial Log  6 h 2 Reduction* 24 h 1 3  7 d 1.0 3 Norecovery 14 d 3.0 No increase No recovery 28 d No increase No increaseNo recovery Fungal Log  7 d No increase 2 Reduction* 14 d No increase 1No increase 28 d No increase No increase No increase *Log₁₀ unitreduction from initial measured inoculum; No increase: not more than 0.5log₁₀ unit increase than previously measured value.

Anti-microbial preservatives can interact with proteins resulting inaggregations and negative effects on stability. Thus, although anecessary component, preservatives pose a significant problem in thedevelopment of stable, multidose formulations of proteins because theytypically induce aggregation of the protein in aqueous solution. Inparticular, increasing or high amounts of preservatives can negativelyimpact the stability of a protein, including effects on physicalstability (aggregation or precipitation) that can impact proteinactivity. For example, to meet the EP preservative efficacyrequirements, relatively high amounts of phenolic compounds, such asphenol or m-cresol, can be required, which can influence stability ofthe protein formulation. For example, preservatives such as phenol,m-cresol, and benzyl alcohol have been shown to induce aggregation ofhuman growth hormone (Maa and Hsu (1996) Int. J. Pharm. 140:155-168),recombinant interleukin-1 receptor (Remmele (1998) Pharm. Res.15:200-208), human insulin-like growth factor I (Fransson (1997) Pharm.Res. 14:606-612), rhIFN-γ (Lam (1997) Pharm. Res. 14:725-729) andcytochrome c (Singh et al. (2011) J. Pharm Sci., 100:1679-89). Thedestabilizing effect that preservatives have on proteins in solution hasbeen a limiting factor in the development of multidose formulations, andto date, most protein therapeutics have been formulated for single useonly.

PH20 hyaluronidase, such as rHuPH20, rapidly loses activity in thepresence of preservatives, likely due to unfolding of the protein andsubsequent aggregate formation. For example, as shown in the Examplesherein, preservatives reduce PH20 enzymatic activity, particularly atelevated temperatures (see also U.S. Provisional Appl. No. 61/520,962;and U.S. application Ser. Nos. 13/507,263 and 13/507,262). For example,following incubation with 0.4% m-cresol for 4 hours, PH20 (e.g.,rHuPH20) retains only about 10% of its activity (see e.g., Example 5).When incubated in the presence of 0.1% phenol and 0.15% or 0.315%m-cresol for 6 days at 37° C., PH20 (e.g., rHuPH20) retains about 0% to15% activity, depending on the presence of other excipients or amountsof other excipients in the formulation (see e.g., Examples 9 and 10).For example, the presence of a higher concentration of salt generallyincreases the stability of PH20. In particular, the melting temperatureof PH20, such as rHuPH20, is reduced significantly when phenolicpreservatives, such as m-Cresol, are added to the formulation. Forexample, the unfolding temperature of rHuPH20 is reduced from 44° C. to24° C. The lower PH20 unfolding temperatures leads to increased PH20aggregation, especially at elevated temperatures, and reduced enzymeactivity. The destabilizing effect is likely due to the hydrophobicnature of the phenolic preservatives. The hydrophobicity of the phenoliccompounds can lead to interaction with rHuPH20 through nonspecificbinding to the protein, ultimately perturbing the structural integrityof rHuPH20. This translates to a significant loss of rHuPH20 enzymaticactivity in the presence of preservatives.

The modified PH20 polypeptides provided herein that exhibit increasedstability in the presence of phenolic preservatives exhibit more than15% enzymatic activity in the presence of at least one phenolicpreservative for at least 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 3 weeks, 4 weeks or more compared to the enzymaticactivity of the modified PH20 polypeptide in the absence of preservativefor the same time period and under the same conditions (except for thepresence of preservative). In some examples, the modified PH20polypeptides provided herein exhibit at least 16%, 17%, 18%, 19%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more enzymatic activity in the presence of a phenolicpreservative compared to in the absence of preservative. For example,the phenolic preservative compound can be phenol, metacresol (m-cresol),benzyl alcohol, and/or parabens including methylparaben orpropylparaben.

In particular examples, the increased stability in the presence ofpreservative is exhibited under temperature conditions of between orabout between 0° C. to 40° C., such as between or about between 2° C. to6° C., 24° C. to 32° C. or 35° C. to 40° C., and generally at or aboutat 4° C. or 5° C., 30° C. or 37° C. It is understood that since hightemperature also can have a destabilizing effect on PH20 activity (seebelow), the percentage of enzymatic activity of a modified PH20polypeptide provided herein in the presence of preservative is greaterat lower temperatures than at higher temperatures.

Generally, the modified PH20 polypeptides provided herein exhibitincreased stability, and the noted enzymatic activities, in the presenceof an anti-microbial effective amount of preservative that kills orinhibits the propagation of microbial organisms in a sample of thecomposition. For example, the modified PH20 polypeptides provided hereinexhibit increased stability in the presence of an anti-microbialeffective amount of preservative that kills or inhibits the propagationof microbial organisms such that at least a 1.0 log₁₀ unit reduction inbacterial organisms occurs at 7 days following inoculation. In someexamples, the modified PH20 polypeptides provided herein exhibitincreased stability in the presence of an anti-microbial effectiveamount of preservative that kills or inhibits the propagation ofmicrobial organisms such that, when tested in an antimicrobialpreservative effectiveness test (APET), following inoculation of thecomposition with a microbial inoculum there is at least a 1.0 log₁₀ unitreduction in bacterial organisms at 7 days following inoculation, atleast a 3.0 log₁₀ unit reduction of bacterial organisms at 14 daysfollowing inoculation, at least no further increase in bacterialorganisms after 28 days following inoculation, and at least no increasein fungal organisms after 7 days following inoculation. In otherexamples, the modified PH20 polypeptides provided herein exhibitincreased stability in the presence of an anti-microbial effectiveamount of preservative that kills or inhibits the propagation ofmicrobial organisms such that, when tested in an antimicrobialpreservative effectiveness test (APET), following inoculation of thecomposition with a microbial inoculum there is at least a 1.0 log₁₀ unitreduction of bacterial organisms at 24 hours following inoculation, atleast a 3.0 log₁₀ unit reduction of bacterial organisms at 7 daysfollowing inoculation, no further increase in bacterial organisms after28 days following inoculation, at least a 1.0 log₁₀ unit reduction offungal organisms at 14 days following inoculation, and at least nofurther increase in fungal organisms after 28 days followinginoculation. In yet another example, the modified PH20 polypeptidesprovided herein exhibit increased stability in the presence of ananti-microbial effective amount of the preservative that kills orinhibits the propagation of microbial organisms such that, when testedin an antimicrobial preservative effectiveness test (APET), followinginoculation of the composition with a microbial inoculum there is atleast a 2.0 log₁₀ unit reduction of bacterial organisms at 6 hoursfollowing inoculation, at least a 3.0 log₁₀ unit reduction of bacterialorganisms at 24 hours following inoculation, no recovery of bacterialorganisms after 28 days following inoculation of the composition withthe microbial inoculum, at least a 2.0 log₁₀ unit reduction of fungalorganisms at 7 days following inoculation, and at least no furtherincrease in fungal organisms after 28 days following inoculation.

For example, the modified PH20 polypeptides provided herein exhibitincreased stability, and above recited enzymatic activity, in thepresence of a total amount of one or more phenolic preservative agentsas a percentage (%) of mass concentration (w/v) that is or is between0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4% inclusive.

Generally, modified PH20 polypeptides provided herein exhibit increasedstability in the presence of m-cresol and/or phenol. For example,modified PH20 polypeptides provided herein exhibit increased stabilityin the presence of m-cresol in an amount as a % of mass concentration(w/v) in a formulation containing the modified PH20 polypeptide ofbetween or about between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%,0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to0.4%. In other examples, modified PH20 polypeptides provided hereinexhibit increased stability in the presence of phenol in an amount at a% of mass concentration (w/v) in a formulation containing the modifiedPH20 polypeptide of between or about between 0.05% to 0.6%, 0.1% to0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2%to 0.3% or 0.3% to 0.4% m-cresol. In further examples, modified PH20polypeptides provided herein exhibit increased stability in the presenceof phenol and m-cresol in an amount as a % of mass concentration (w/v)in a formulation containing the modified PH20 polypeptide of between orabout between 0.05% to 0.25% phenol and between or about between 0.05%to 0.3% m-cresol, between or about between 0.10% to 0.2% phenol andbetween or about between 0.6% to 0.18% m-cresol, between or aboutbetween 0.1% to 0.15% phenol and 0.8% to 0.15% m-cresol, between orabout between 0.10% to 0.15% phenol and between or about between 0.06%to 0.09% m-cresol, or between or about between 0.12% to 0.18% phenol andbetween or about between 0.14% to 0.22% m-cresol.

In examples herein, modified PH20 polypeptides exhibit more than 15%,such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymaticactivity in the presence of at least about between or between 0.3% to0.4%, inclusive, m-cresol and/or phenol for at least 4 hours at 37° C.compared to the enzymatic activity of the modified PH20 polypeptide inthe absence of the preservative for the same time period and under thesame conditions (except for the presence of preservative). For example,modified PH20 polypeptides exhibit more than 15%, such as at least 16%,17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presenceof about or 0.4% m-cresol for at least 4 hours at 37° C. compared to theenzymatic activity of the modified PH20 polypeptide in the absence ofthe preservative for the same time period and under the same conditions(except for the presence of preservative). Modified PH20 polypeptidesprovided herein also exhibit more than 15%, such as at least 16%, 17%,18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presence of atleast about between or between 0.2% to 0.4%, inclusive, m-cresol and/orphenol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days at37° C. compared to the enzymatic activity of the modified PH20polypeptide in the absence of preservative for the same time period andunder the same conditions (except for the presence of preservative). Forexample, modified PH20 polypeptides provided herein exhibit more than15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymaticactivity in the presence of about or 0.10% phenol and about or 0.15%m-cresol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days at37° C. compared to the enzymatic activity of the modified PH20polypeptide in the absence of preservative for the same time period andunder the same conditions (except for the presence of preservative). Inother examples, modified PH20 polypeptides provided herein exhibit morethan 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or moreenzymatic activity in the presence of about or 0.315% m-cresol for atleast 1 day, 2 days, 3 days, 4 days, 5 days or 6 days, generally for atleast 6 days, at 37° C. compared to the enzymatic activity of themodified PH20 polypeptide in the absence of preservative for the sametime period and under the same conditions (except for the presence ofpreservative).

For example, such modified PH20 polypeptides provided herein thatexhibit increased stability to phenol compounds contain an amino acidreplacement (substitution) at one or more amino acid positionscorresponding to positions 10, 12, 20, 22, 26, 34, 36, 46, 50, 52, 58,68, 70, 74, 82, 83, 84, 86, 97, 127, 131, 138, 142, 143, 144, 166, 169,174, 193, 195, 196, 204, 205, 206, 213, 219, 234, 237, 238, 240, 249,261, 267, 277, 279, 291, 309, 310, 314, 315, 317, 318, 347, 367, 375,376, 399, 401, 407, 416, 419, 421, 431, 433, 439, 440, 443 or 445 withreference to amino acid positions set forth in SEQ ID NO:3. For example,the amino acid positions can be replacements at one or more positionscorresponding to replacement of (P) at position 10 (P10), V12, A20, S22,L26, D34, S36, 146, G50, G52, V58, D68, I70, T74, K82, 183, S84, Q86,T97, D127, N131, Q138, V142, Q143, L144, V166, 1169, L174, H193, K195,K196, F204, N205, V206, D213, N219, Q234, V237, A238, T240, E249, S261,A267, V277K279, G291, 1309, M310, K314, S315, L317, Q347, P367, E375,K376, Y399, S401, S407, D416, A419, D421, D431, F433, E439, T440, P443or 1445 with reference to amino acid positions set forth in SEQ ID NO:3.

Exemplary of amino acid replacements in the modified PH20 polypeptidesprovided herein include, but are not limited to, replacement with:glycine (G) at a position corresponding to position 10; K at a positioncorresponding to position 12; S at a position corresponding to position20; T at a position corresponding to position 22; M at a positioncorresponding to position 26; W at a position corresponding to position34; N at a position corresponding to position 36; L at a positioncorresponding to position 46; M at a position corresponding to position50; T at a position corresponding to position 52; S at a positioncorresponding to position 52; C at a position corresponding to position58; K at a position corresponding to position 58; Rat a positioncorresponding to position 58; N at a position corresponding to position58; Y at a position corresponding to position 58; P at a positioncorresponding to position 58; H at a position corresponding to position58; P at a position corresponding to position 68; V at a positioncorresponding to position 70; E at a position corresponding to position74; L at a position corresponding to position 82; N at a positioncorresponding to position 82; V at a position corresponding to position83; Q at a position corresponding to position 83; S at a positioncorresponding to position 83; G at a position corresponding to position83; N at a position corresponding to position 84; A at a positioncorresponding to position 86; K at a position corresponding to position86; E at a position corresponding to position 97; L at a positioncorresponding to position 97; R at a position corresponding to position127; Rat a position corresponding to position 131; L at a positioncorresponding to position 138; K at a position corresponding to position142; N at a position corresponding to position 142; P at a positioncorresponding to position 142; S at a position corresponding to position142; T at a position corresponding to position 142; G at a positioncorresponding to position 143; K at a position corresponding to position143; T at a position corresponding to position 144; Q at a positioncorresponding to position 166; T at a position corresponding to position166; L at a position corresponding to position 169; G at a positioncorresponding to position 174; N at a position corresponding to position174; Q at a position corresponding to position 193; T at a positioncorresponding to position 195; N at a position corresponding to position195; E at a position corresponding to position 196; Rat a positioncorresponding to position 196; P at a position corresponding to position204; A at a position corresponding to position 205; E at a positioncorresponding to position 205; I at a position corresponding to position206; A at a position corresponding to position 213; I at a positioncorresponding to position 219; M at a position corresponding to position234; T at a position corresponding to position 237; H at a positioncorresponding to position 238; Q at a position corresponding to position240; V at a position corresponding to position 249; A at a positioncorresponding to position 261; K at a position corresponding to position261; Tat a position corresponding to position 267; K at a positioncorresponding to position 277; H at a position corresponding to position279; V at a position corresponding to position 279; V at a positioncorresponding to position 291; E at a position corresponding to position309; Q at a position corresponding to position 310; Y at a positioncorresponding to position 314; Y at a position corresponding to position315; N at a position corresponding to position 317; W at a positioncorresponding to position 317; D at a position corresponding to position318; G at a position corresponding to position 347; A at a positioncorresponding to position 367; R at a position corresponding to position375; Rat a position corresponding to position 376; V at a positioncorresponding to position 399; E at a position corresponding to position401; A at a position corresponding to position 407; L at a positioncorresponding to position 416; K at a position corresponding to position419; H at a position corresponding to position 421; E at a positioncorresponding to position 431; Tat a position corresponding to position433; V at a position corresponding to position 433; C at a positioncorresponding to position 439; P at a position corresponding to position440; G at a position corresponding to position 443; N at a positioncorresponding to position 445, each with reference to amino acid residuepositions set forth in SEQ ID NO:3.

The amino acid replacement(s) can be in a PH20 polypeptide as set forthin any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or avariant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity thereto. For example, the replacement(s) can be in ahuman PH20 polypeptide, for example, any set forth in any of SEQ ID NOs:3, 7, 32-66, 69 or 72 or a variant thereof.

Exemplary modified PH20 polypeptides that exhibit increased stability tophenol compounds compared to the unmodified PH20 polypeptide (e.g., setforth in SEQ ID NO:3) are any having the sequence of amino acids setforth in any of SEQ ID NOs: 83, 88, 93, 94, 101, 144, 148, 158, 171,176, 175, 177, 178, 180, 182, 183, 184, 185, 194, 221, 240, 259, 260,261, 262, 263, 264, 268, 270, 272, 307, 309, 327, 334, 341, 351, 352,353, 356, 357, 358, 359, 361, 424, 426, 430, 434, 436, 443, 444, 445,446, 447, 449, 450, 451, 454, 461, 467, 480, 487, 489, 492, 495, 504,505, 509, 527, 544, 576, 589, 600, 603, 607, 612, 614, 647, 658, 683,687, 733, 736, 741, 754, 763, 768, 781, 796, 797, 809, 818, 829 or 837or having a sequence of amino acids that exhibits at least 68%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 83, 88,93, 94, 101, 144, 148, 158, 171, 176, 175, 177, 178, 180, 182, 183, 184,185, 194, 221, 240, 259, 260, 261, 262, 263, 264, 268, 270, 272, 307,309, 327, 334, 341, 351, 352, 353, 356, 357, 358, 359, 361, 424, 426,430, 434, 436, 443, 444, 445, 446, 447, 449, 450, 451, 454, 461, 467,480, 487, 489, 492, 495, 504, 505, 509, 527, 544, 576, 589, 600, 603,607, 612, 614, 647, 658, 683, 687, 733, 736, 741, 754, 763, 768, 781,796, 797, 809, 818, 829 or 837 and contains the amino acid replacement,exhibits hyaluronidase activity and exhibits increased stability in thepresence phenol compounds compared to the corresponding unmodifiedpolypeptide.

In particular, provided herein is a modified PH20 polypeptide thatcontains an amino acid replacement with P at a position corresponding toamino acid residue 204 with reference to SEQ ID NO:3. Typically, themodified PH20 polypeptide is a human polypeptide. For example, providedherein is a modified PH20 polypeptide that contains an amino acidreplacement F204P in a sequence of amino acids set forth in any of SEQID NOs: 3, 7, 69, 72 or 32-66, or a sequence of amino acids thatexhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to any of SEQ ID NOs:3, 7, 69, 72 or32-66 so long as the modified polypeptide contains the amino acidreplacement corresponding to F204P. In other cases, the modified PH20polypeptide is a non-human polypeptide. For example, provided herein isa modified PH20 polypeptide that contains an amino acid replacementF204P in a sequence of amino acids set forth in SEQ ID NO:10, 12, 14,857, 859, 861 or 870 or a sequence that exhibits at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any of SEQ ID NOs: 10, 12, 14, 857, 859, 861 or 870 so longas the modified polypeptide contains the amino acid replacementcorresponding to F204P. In a further example, provided herein is amodified PH20 polypeptide that contains an amino acid replacement F205Pin a sequence of amino acids set forth in SEQ ID NO:24 or Y204P in SEQID NO:31, or a sequence that exhibits at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQID NO:24 or 31. Exemplary of such a modified PH20 polypeptide is apolypeptide having the sequence of amino acids set forth in SEQ IDNO:449, or having a sequence of amino acids that exhibits at least 68%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:449 andcontains the amino acid replacement F204P, exhibits increasedhyaluronidase activity and exhibits increased stability to phenolcompounds compared to the corresponding unmodified polypeptide (e.g.,SEQ ID NO:3). In any of the above examples, the modified PH20polypeptide that contains an amino acid replacement with P at a positioncorresponding to amino acid residue 204 with reference to SEQ ID NO:3does not have the sequence of amino acids set forth in SEQ ID NO:15-22,28 or 29.

In another example, provided herein is a modified PH20 polypeptide thatcontains an amino acid replacement at a position corresponding to aminoacid residue 58 with reference to SEQ ID NO:3. Exemplary of amino acidreplacements are replacement with lysine (K) or with arginine (R) at aposition corresponding to amino acid residue 58 with reference to SEQ IDNO:3. Typically, the modified PH20 polypeptide is a human polypeptide.For example, provided herein is a modified PH20 polypeptide thatcontains an amino acid replacement V58K or V58R in a sequence of aminoacids set forth in any of SEQ ID NOs: 3, 7, 69, 72 or 32-66, or asequence of amino acids that exhibits at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to anyof SEQ ID NOs:3, 7, 69, 72 or 32-66. In other cases, the modified PH20polypeptide is a non-human polypeptide. For example, provided herein isa modified PH20 polypeptide that contains an amino acid replacement V58Kor V58R in a sequence of amino acids set forth in SEQ ID NOs:10, 12, 14,20, 22, 24, 29, 857, 859, 861 or 870 or a sequence that exhibits atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to any of SEQ ID NOs: 10, 12, 14, 20, 22, 24, 29,857, 859, 861 or 870. In a further example, provided herein is amodified PH20 polypeptide that contains an amino acid replacement A58Rin a sequence of amino acids set forth in SEQ ID NO:16 or 31, or asequence that exhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:16 or 31.Exemplary of such a modified PH20 polypeptide is a polypeptide havingthe sequence of amino acids set forth in SEQ ID NO:182, or having asequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO:182, which contains the amino acidreplacement V58R and exhibits increased hyaluronidase activity andexhibits increased stability in the presence of phenol compoundscompared to the corresponding unmodified polypeptide (e.g., SEQ IDNO:3).

ii. Thermophiles

At elevated temperatures, PH20 hyaluronidases can lose activity.

Provided herein are modified PH20 polypeptides that exhibit increasedstability at elevated temperatures of between or about between 30° C. to45° C., inclusive, such as between or about between 35° C. to 42° C., inparticular at or about 37° C. For example, provided herein are modifiedPH20 polypeptides that are stable at elevated temperatures greater than32° C. such as 35° C. to 45° C., 37° C. to 42° C. and in particular ator about 37° C. for at least 3 hours, 4 hours, 5 hours, 6 hours, 12hours, 1 day, 2 days, 3 days, 4 days, at least 5 days, at least 6 daysor at least 7 days. Modified PH20 polypeptides that exhibit stability atelevated temperatures can be used in applications where temperatures areelevated, can fluctuate or can increase. This can occur, for example, inmethods of administration utilizing pumps or other continuous infusiondevices.

In particular, modified PH20 polypeptides provided herein that exhibitstability at elevated temperatures exhibit increased hyaluronidaseactivity at elevated temperature compared to the corresponding PH20polypeptide not containing the modification, e.g., amino acidreplacement. The PH20 polypeptides can exhibit increased hyaluronidaseactivity upon incubation at elevated temperatures greater than 32° C.such as 35° C. to 45° C. or 37° C. to 42° C., in particular at or about37° C. for at least 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days,3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4days, at least 5 days, at least 6 days or at least 7 days compared tothe corresponding PH20 polypeptide not containing the modificationincubated under the same conditions. For example, the hyaluronidaseactivity can be increased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to thecorresponding PH20 polypeptide not containing the modification incubatedunder the same conditions. For example, the hyaluronidase activity canbe increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-foldor more compared to the corresponding PH20 polypeptide not containingthe modification incubated under the same conditions.

In other examples, modified PH20 polypeptides provided herein thatexhibit stability at elevated temperatures retain hyaluronidase activityat elevated temperatures compared to the activity of the modified PH20polypeptide incubated at non-elevated temperatures under the sameconditions (except for the differences in temperature). For example,modified PH20 polypeptides exhibit greater than or about 50%, such asgreater than or at least 55%, 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% of the activity at elevatedtemperatures greater than 32° C. such as 35° C. to 45° C. or 37° C. to42° C., in particular at or about 37° C. compared to the activity of thePH20 at non-elevated temperatures of between or about between 2° C. to8° C. In some examples, modified PH20 polypeptides provided herein thatexhibit stability at elevated temperatures exhibit increased activity atelevated temperatures compared to the activity of the modified PH20polypeptide incubated at non-elevated temperatures under the sameconditions (except for the difference in temperature). For example,modified PH20 polypeptides exhibit greater than or about 10% increasedactivity, such as greater than or at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 400%, 500% or more of activity at elevatedtemperatures greater than 32° C. such as 35° C. to 45° C. or 37° C. to42° C., in particular at or about 37° C. compared to the activity of thePH20 at non-elevated temperatures of between or about between 2° C. to8° C. For example, modified PH20 polypeptides exhibit greater than or atleast about 1.1-fold the hyaluronidase activity, such as greater than orat least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold or more of activityat elevated temperatures greater than 32° C. such as 35° C. to 45° C. or37° C. to 42° C., in particular at or about 37° C. compared to theactivity of the PH20 at non-elevated temperatures of between or aboutbetween 2° C. to 8° C.

For example, such modified PH20 polypeptides provided herein thatexhibit increased stability at elevated temperatures contain an aminoacid replacement (substitution) at one or more amino acid positionscorresponding to positions 1, 11, 12, 14, 20, 26, 29, 34, 50, 58, 70,82, 83, 84, 86, 87, 140, 142, 143, 147, 152, 166, 167, 172, 174, 178,193, 195, 206, 212, 213, 219, 233, 237, 240, 267, 277, 291, 292, 309,313, 314, 317, 318, 347, 367, 368, 371, 374, 389, 392, 395, 396, 406,419, 421, 439 or 443 with reference to amino acid positions set forth inSEQ ID NO:3. For example, the amino acid positions can be replacementsat one or more positions corresponding to replacement of (L) at position1 (L1), N11, V12, F14, A20, L26, F29, D34, G50, V58, I70, K82, 183, S84,Q86, D87, Q140, V142, Q143, T147, K152, V166, E167, G172, L174, N178,H193, K195, V206, D212, D213, N219, Q233, V237, T240, A267, V277, G291,E292, 1309, M313, K314, L317, L318, Q347, P367, D368, A371, L374, E389,E392, S395, E396, L406, A419, D421, E439 or P443, with reference toamino acid positions set forth in SEQ ID NO:3. The resulting modifiedPH20 polypeptide exhibits increased stability at elevated temperaturesgreater than 32° C. such as 35° C. to 45° C., 37° C. to 42° C. and inparticular at or about 37° C. for at least 3 hours, 4 hours, 5 hours, 6hours, 12 hours, 1 day, 2 days, 3 days, 4 days, at least 5 days, atleast 6 days, at least 7 days or more.

Exemplary amino acid replacements in the modified PH20 polypeptidesprovided herein include, but are not limited, replacement with: R at aposition corresponding to position 1; S at a position corresponding toposition 11; I at a position corresponding to position 12; V at aposition corresponding to position 14; S at a position corresponding toposition 20; M at a position corresponding to position 26; with R at aposition corresponding to position 29; W at a position corresponding toposition 34; M at a position corresponding to position 50; K at aposition corresponding to position 58; Q at a position corresponding toposition 58; Q at a position corresponding to position 58; V at aposition corresponding to position 70; L at a position corresponding toposition 82; Q at a position corresponding to position 83; Rat aposition corresponding to position 84; A at a position corresponding toposition 86; S at a position corresponding to position 87; K at aposition corresponding to position 140; S at a position corresponding toposition 142; T at a position corresponding to position 142; K at aposition corresponding to position 143; S at a position corresponding toposition 147; T at a position corresponding to position 152; T at aposition corresponding to position 166; D at a position corresponding toposition 167; A at a position corresponding to position 172; G at aposition corresponding to position 174; N at a position corresponding toposition 174; Rat a position corresponding to position 178; Q at aposition corresponding to position 193; T at a position corresponding toposition 195; I at a position corresponding to position 206; S at aposition corresponding to position 212; A at a position corresponding toposition 213; I at a position corresponding to position 219; G at aposition corresponding to position 233; T at a position corresponding toposition 237; A at a position corresponding to position 240; Q at aposition corresponding to position 240; T at a position corresponding toposition 267; E at a position corresponding to position 277; S at aposition corresponding to position 291; H at a position corresponding toposition 292; V at a position corresponding to position 292; S at aposition corresponding to position 309; H at a position corresponding toposition 313; S at a position corresponding to position 314; I at aposition corresponding to position 317; Tat a position corresponding toposition 317; W at a position corresponding to position 317; Rat aposition corresponding to position 318; G at a position corresponding toposition 347; A at a position corresponding to position 367; R at aposition corresponding to position 368; S at a position corresponding toposition 371; P at a position corresponding to position 374; A at aposition corresponding to position 389; V at a position corresponding toposition 392; A at a position corresponding to position 395; H at aposition corresponding to position 396; N at a position corresponding toposition 406; H at a position corresponding to position 419; K at aposition corresponding to position 419; Rat a position corresponding toposition 421; S at a position corresponding to position 421; A at aposition corresponding to position 439; C at a position corresponding toposition 439; or G at a position corresponding to position 443, eachwith reference to amino acid residue positions set forth in SEQ ID NO:3.

The amino acid replacement(s) can be in a PH20 polypeptide as set forthin any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or avariant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity thereto. For example, the replacement(s) can be in ahuman PH20 polypeptide, for example, any set forth in any of SEQ ID NOs:3, 7, 32-66, 69 or 72 or a variant thereof.

Exemplary modified PH20 polypeptides that exhibit increased stability tophenol compounds compared to the unmodified PH20 polypeptide (e.g., setforth in SEQ ID NO:3) are any having the sequence of amino acids setforth in any of SEQ ID NOs: 79, 85, 87, 90, 93, 101, 114, 144, 171, 178,181, 221, 259, 262, 269, 270, 282, 343, 356, 357, 359, 368, 395, 426,429, 432, 434, 436, 441, 443, 444, 454, 460, 461, 467, 477, 487, 491,492, 509, 525, 550, 554, 557, 584, 593, 599, 605, 611, 612, 617, 647,658, 667, 676, 679, 709, 720, 723, 727, 740, 761, 763, 772, 773, 808,809, or 829 or having a sequence of amino acids that exhibits at least68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:79, 85, 87, 90, 93, 101, 114, 144, 171, 178, 181, 221, 259, 262, 269,270, 282, 343, 356, 357, 359, 368, 395, 426, 429, 432, 434, 436, 441,443, 444, 454, 460, 461, 467, 477, 487, 491, 492, 509, 525, 550, 554,557, 584, 593, 599, 605, 611, 612, 617, 647, 658, 667, 676, 679, 709,720, 723, 727, 740, 761, 763, 772, 773, 808, 809, or 829 and containsthe amino acid replacement, exhibits hyaluronidase activity and exhibitsincreased stability to elevated temperatures compared to thecorresponding unmodified polypeptide.

iii. Absence of Salt

PH20 denatures in the presence of low salt or no salt. Thus, PH20requires a high salt concentration of between or about between 140 mM to200 mM to maintain stability. Other therapeutic agents, for exampleinsulin, exhibit decreased solubility and increasedcrystallization/aggregation in the presence of high salt. Thus, the highsalt requirements of PH20 can affect the solubility and/or activity ofco-formulated therapeutic agents, while the presence of low salt candecrease the activity of PH20. This can create problems for generatingPH20 co-formulations.

Provided herein are modified PH20 polypeptides that exhibit increasedstability in the presence of low concentrations of salt (e.g. NaCl) lessthan 100 mM, for example, less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM,40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. Generally, themodified PH20 polypeptides provided herein exhibit stability in thepresence of low concentrations of salt, for example, low concentrationsof NaCl of between or about between 10 mM NaCl and 100 mM NaCl, such asbetween or about between 15 mM to 80 mM NaCl. The modified PH20polypeptides provided herein that exhibit stability at lowconcentrations of salt, such as low concentrations of NaCl (i.e., lessthan 100 mM or less), exhibit increased hyaluronidase activity comparedto the corresponding PH20 not containing the modification(s) (e.g.,amino acid replacements). For example, modified PH20 polypeptidesexhibit greater than or about 10% increased activity, such as greaterthan or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500% or more of activity at low concentrations of salt, suchas low concentrations of NaCl (i.e., less than 100 mM), compared to theactivity of the corresponding PH20 not containing the amino acidmodification(s) (e.g., amino acid replacement(s) under the sameconditions). For example, modified PH20 polypeptides exhibit greaterthan or at least about 1.1-fold the hyaluronidase activity, such asgreater than or at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-foldor more of activity at low concentrations of NaCl less than 100 mMcompared to the activity of the corresponding PH20 not containing theamino acid modification(s) (e.g., amino acid replacement(s) under thesame conditions.

2. Inactive Mutants

Provided herein are modified PH20 polypeptides that contain one or moreamino acid replacements in a PH20 polypeptide and that are inactive,whereby the polypeptides do not exhibit hyaluronidase activity orexhibit low or diminished hyaluronidase activity. The modified PH20polypeptides provided herein that are inactive generally exhibit lessthan 20%, such as less than 10%, of the hyaluronidase activity of awildtype or reference PH20 polypeptide, such as the polypeptide setforth in SEQ ID NO: 3 or 7. For example, modified PH20 polypeptidesprovided herein that are inactive exhibit less than 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.05% or less of the hyaluronidase activity of a wildtype or referencePH20 polypeptide, such as the corresponding polypeptide not containingthe amino acid modification (e.g., amino acid replacement), for example,a polypeptide set forth in SEQ ID NO:3 or 7.

For example, provided herein are PH20 polypeptides that are inactive andthat are modified, for example by amino acid replacement orsubstitution, compared to a wildtype or reference PH20 polypeptide. Forexample, a modified PH20 polypeptide provided herein that is inactivecontains one or more amino acid replacements at position(s)corresponding to position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 27, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94,95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,143, 144, 145, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 161,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 331, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 408, 410, 411, 412, 413, 414,415, 416, 417, 419, 420, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 434, 437, 438, 439, 440, 441, 442, 443, 444, or 447 withreference to amino acid positions set forth in SEQ ID NO:3, so long asthe resulting modified PH20 polypeptide is inactive and exhibits lessthan 20%, and generally less than 10%, of the hyaluronidase activity ofthe corresponding PH20 polypeptide not containing the amino acidreplacement. Typically, the amino acid residue that is modified (e.g.,replaced) at the position corresponding to any of the above positions ina PH20 polypeptide is an identical residue, a conservative residue or asemi-conservative amino acid residue to the amino acid residue set forthin SEQ ID NO:3.

Exemplary amino acid replacements at any of the above correspondingpositions are set forth in Table 5. Reference to corresponding positionin Table 5 is with reference to positions set forth in SEQ ID NO:3. Itis understood that the replacements can be made in the correspondingposition in another PH20 polypeptide by alignment therewith with thesequence set forth in SEQ ID NO:3 (see e.g., FIGS. 1 and 2 ), wherebythe corresponding position is the aligned position. The amino acidreplacement(s) can be at the corresponding position in a PH20polypeptide as set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72,856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, so long as theresulting modified PH20 polypeptide is inactive. For example, thereplacement(s) can be in a corresponding position in a human PH20polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7,32-66, 69 or 72, or a variant thereof. In particular, any one or more ofthe replacements are in SEQ ID NO:3, so long as the resulting modifiedPH20 polypeptide is inactive and exhibits less than 20%, and generallyless than 10%, of the hyaluronidase activity of the PH20 polypeptide setforth in SEQ ID NO:3

TABLE 5 Inactive Mutants Corresponding Corresponding CorrespondingPosition Replacement Position Replacement Position Replacement 2 H K W Y3 A G K P T V 4 D E F G L P W Y 5 D G I L M N P Q R 6 E F T V Y 7C D F G H I K L Q T V W Y R S T W Y 8 D E G H N R S W 9 C D E G N P 10F I L M Y 11 A C F I L P T W Y 12 G H W 13 E G I L M V 14A E G H K N P Q W 15 E F G K N P Q R S 16 A C D E F G H K Y M P R S T Y17 D E G H I L N P Q 18 C D F G H I L M P 19 A C F G H I L M PR S T V W Y Q S T V Y Q R S V W Y  20 D E F H K L N P R 21A C D E G H I L M 22 C E G K P T V Y R S T V W 23 A F L M N P R S T 25D E F G H I K L N 27 C V P R S T V Y 33 C D H N V Y 34 I L N S T V 35A D G P R S 36 C F V W Y 37 C E G N S 38 E G K L N Q R T W 39 C D F W 40A D E G K N R S T 41 Q V 42 D E H I K L M P Q 43 A E F G I K L Q R 44A C F G H I L N Q R S T V V R S T W Y 45 A D F G P W 46 P W 47 V 48 P 49C D G H P 50 V 51 C F I M P T W Y 52 C E F W Y 53 A C D E G H L N PQ R S T W Y 54 D E G P R Y 55 A D G H N P Q R T 56 A C E G H I K L P V YR S T V W 57 A D F G I M P Q R 58 A 59 A E I L M P R T V V W W Y 60A D F G H I L N P 61 A E F G H N P Q R 62 A C D F I K L M P Q S T V YT W Y Q R S T V Y 63 C G P 64 A C D E F G H I K 65 A C D G H I K N RL P Q R S T V W S T V W Y 66 A C D E G I K L N 67 D E G P R T W 68A C G I L P V Y P S T V 69 N T 70 Q 71 P 72 C F H I P V W 73 P 75 D G P76 A C F G I K L P Q 77 D E L P Q R T V 78 A D I M P T Y R S T V W 79A D F G H K N P S 80 A D E F G I K L M  81 A C E G H L N P S W YN R S T V Y V W Y 82 Y E K 84 Y 85 A C D E F G H N Q S T 86 C P 87 P 88A C E F G I K L M P R S T V Y 89 A D E G Q S T W Y 90 C G 91D E F G H I L T 92 E F H K P Q R W Y 94 G P 95 A C E F G H K LM P Q S V W Y  96 S V H P R S T W 98 P 99 C E G I N P V W 100C E F G N P R S T 101 A C F H I K L M N 102 P W Y Q R S T 103A E F G H I L Q R T 104 F P W 105 C M N V W Y 106 A C D F H L M N P 107A C H K P Q S V W 108 D E F K L M P Q T S W Y V Y 109 C D E L M R T W110 F K L M P W 111 H I Q 112 C E G H L N P S 113 R V 114 I L P T V 115A C D F G H I K L 116 A C D E G H I L N 117 D G I K N Q R S V M R S V YP Q S V W  W 118 C D E G P R W Y  119 A K I L N P R 121 A C E F G H K LM P W Y 122 A C E F I K Q R S 123 A C D E H L M P Q 124 C D E F N T VR S T V Y 125 C D G L N W  126 F H I L N P Y 127 K 128 E P 129A C D E G H L P Q 130 C D G H L N S T S T V W W Y 131 P 132 P 133D E F G H L M N P R T V W 134 A C D F G H K P Q 135 P 136 P R S W 137F G H N P R W Y 138 V 139 P 143 C H P R S T  144 A E F I K P Q S V 145T W Y 149 E 149 P 150 V 152 L 153 E F M P R T V 154 D E G P S W Y 155P Y  156 P 157 A C D E G H I K L M P Q R S T V 158 D K P R Y 159 W Y 161W 163 C P 164 A C D E G H N P Q 165 C H P T R 166 D 167 V 168A C D E F G K L P R S V W Y  169 A D F G H K N P Q 170 C D E G M P W Y171 C D H M N R S W S T Y Y 172 D E I L P Q T V W 173 D E G H I L M P S174 P Y V W Y 175 C D G K P R S 176 A C E F G H I P Q 177A C D F G H L M S T V W Q R S T V W 178 E I L V W 180 A C E P R S 181A C D E F H I K L R S 182 A C D E H N P Q R 183 C D E G I K N P Q 184A C D E F G H K L S T V Y R S V M P R S V 185 A D E F G I K P R S 186A D G H I K L N P  187 A F G H I L M N Q T V W Y Q R S V W R S T V W Y188 A C F G H L M N P 189 A E G H K L M N P 190 C E F G H K L N QQ R S T V W R S T V W Y R S T V W 191 A E F G K L M P Q 192C F G K L M N P Q 193 A D K L M P V R S T V W Y R V W Y  194A C I L P S T V 195 S 197 C 198 V W 199 E G H I K L P R S 200A F G H K L M P W Q R S W Y 201 A F L M N P R S T 202 A E F G H K N P Q203 A D E G H L M N  V W R V W Y  Q R S T V 204 A C E G H I K Q R 206C D F G P Y  207 A F G M P Q R S T S T V W 208 D G P W 209 C P 210A C D E G K M N P S T V W Y 211 C F G H I K M P R 212 A G H I K L M P V213 P S S T V W W 214 A C D E G H K N P 215 C P 216 D E G H I K L M NR S T Y P Q R T V 217 A C G H P Q S T V 218 A I K L P S V 219 P W 220G K N P R W 221 D E H K P R 222 P Y 223 C D E G H K L P Q 224A D E F G M P Q R 225 A D E G H K P Q R S T V W Y  S T W Y R T V W 226A C D E F G L N Q 227 A F G H I K L M P 228 A E F G H L M N PR S T V W Y Q R T V W Y  R S T W 229 E F G K L P Q T V 230A E G H K M N P R 231 A C D F G H I K L W S T V W Y P Q R S V 232C G H K L N P Q V 233 D I P S T 234 A D E G H N P S T Y V W 235F L M R W Y 236 C I L N Q T Y 238 F G L P V W Y 239 C F G H I L P R S T240 E F G N W Y  241 A C D E G I P R S V W Y T V W 242 A C D G I L M P R243 C D F G H L M P Q 244 A D G I V Y S T V W  R S W Y 245A C F L P Q R S T 246 A C D E G H I K L 247 A C F H N P Q R S VM P S T V W T W Y 248 C D E G I M P T  249 A G H I K M Q S Y 250C F G H K L M N P Q R S T V W 251 D F G H K P S T W 252A D E F G H I K L 253 A D E G H L M N N P S T Y Q R S W 254C D E G I K L P Q 255 C D L P V W 256 C D E G [ R T V W Y 257 D 258L P V W 260 C P 261 P 262 A D E G H I K Q R 263 E F P Q W S T V W Y 264D E F G L M R T V 265 A D F G H K L M 266 A C G H M P Q R W Y N Q R SS T V W 267 D G H I K N R S W 268 A C F G H K L N P 269 E K L M N P Q RQ S T V W  270 A C E F G H I P Y 271 A D E H K T W 272 H L N P W 273A C D G I L P Q S 274 C E G H N Q W Y 275 A F G I K L M Q T V W V W 276F P W 278 M P 279 A C F G L W Y 280 D I M N R S T V W 281A D G H I K N P Q 282 F L V W Y R S V W 283 A C D F W 284 C D F W 284C I P 285 K P R T V 286 A C D F H K M P T 287 A C D E G K L N P Y Q R S288 D E F G H I K P R T 289 A C E G H L P Q R 290 D Q Y S Y 291A C D E F M N T W 292 I L T 293 E N Y 294 A E G H K L N P Q 295C G H I L N P T V 296 C F G I K M Q R S R S T W Y T V W Y  297C E H L N P Q R S 298 C E L M N P Q S T 299 A C D F G H L M T Y W YP Q T 300 A C D E F L M N P 301 E G H K M N P Q R 302 C D E F G H L M PQ S T V W  S W Y R S T Y 303 A C D E F G K L M 304 A C D G I M N P Q 305L P Q R S T V Y R W Y S T V Y 306 A C H I L V W Y 307 C I P 308C F L M V W Y 310 C E F K L 311 C E F I L P V W 312 C E M V W 313 C 314C L W 315 C I V 316 E G I K L M P R S T 317 G P 318 C P W V W Y 319C E F G H I K M P 320 C P V 321 E M P Q R S V W Y 322C D E G I L N P R S 323 A C E G H K N R S 324 C F P V W Y T V W T V 325C R E G H N W 327 A E F G H N Q R S 329 C F G H I K L N Q T V W Y R S T V W Y 330 A C D E G I L M N 331 A C D E F H K Q R 332A C D E F G H K L P R S V W S T W Y N P R S T Y 333 G H I K P R S T W334 A C D E G M N R S 335 F G H I K L P V W Y Y 336 A E F G K N P R S337 C F G I K L M R T 338 C D E F G H I K L T V W Y W P R T V 339D E F G H L N P S 340 A C D E F G H K P 341 A E G H K L M N T V W YR S T V W Q R S T V Y 342 D E F H K L M P Q 343 C D F I P W 344F G H L M N P Q R T Y R S T W Y 345 A C E H K N Q R T 346A D F G I K L M P 347 C F I P T V W V Y R S T V W 348 C H I L P Q R T V349 D F G P V W Y 350 A D E F H K L M W Y N P R S T V Y 351C D E F H N R W Y 352 A D E F G K M P Q 353 C F G H K L M Q R S T V W YR S W 354 C D E G H I K L M 355 D F G H L M N P Q 356 C G K L P R T VP Q S V W Y  R S T V W Y W 357 D E F G L M Q R 358 E H I K P Q R W 359A F G L P W 360 A C E F G I K L M  361 A C E G M N P Q R 362A C E G H K L M P Q R V S V W N P R S T V W 363 A C D E F G H I P 364A C D E F G K L M 365 A C D E G M N P Q R S T V W  P R S T V YQ R S T W Y 366 A C E F G K M P Q 367 E F I L M Q V 368 C P W R T W 369C E F I K L P Q V 370 A D E G H K L N P 371 P W Q R S V Y 372A D E F G H K L N 373 C P W 374 D E P R S T V W 375 C F P V Y 376 I P W377 C I L V 378 D E F I L M Q T W 379 A C E F I L M W  380 C D E G Q R SY 381 G L P W Y 382 E G H K L M N P Q 383 G P R S T W Y  384 C F M Q S T385 C L M P W Y 386 A C F G H I L M N Q R S T V Y 387 C E F G H I L M N388 C G P Q 389 F V V W Y 390 A C E F G H L N P 391 A D G H K N P Q R392 C P R S T V W Y S T V W Y 393 C P 394 A D E G I K N P Q 395 C ; , [R S T V 396 C F G I P Y 397 A C E F G I L M P 398 A C E G H I L N PQ T V R S T V W Y  399 D P 400 A D E F G I L M P 401 C F H K R W YQ R S T V Y 402 A D E F L M P Q R 403 A C E G H K L M 404C D F G H L M N S T V W Y  N P Q R T R V W Y 405 C I V 406 P R 408A E F G I K L P R S T V W Y 410 W 411 D E F G 412 E H 413 H I K L P 414A D E G H K R S T 415 C D E P 416 C S 417 A D E F G H K M P 419 D P Q R420 A D F G H K L N R 422 C D G H L M N Q 423 A D E F G H L M P S T W YR S Y Q R S T V W 424 A C E G H N Q R S 425 E L P W Y 426 C F M R W Y427 A C F L P V W Y 428 A C D E G H N R S 429 A D K L N P S T V Y W Y430 A D E L M N S T V 431 P 432 C F I K L M P Y 434 H K P Q R W 437 T438 Y 439 N R 440 Q 441 R 442 M N S 443 D

3. Additional Modifications and Conjugates

The modified PH20 polypeptides include those that contain chemical orposttranslational modifications. In some examples, modified PH20polypeptides provided herein do not contain chemical orposttranslational modifications. Chemical and post-translationalmodifications include, but are not limited to, PEGylation, sialation,albumination, glycosylation, farnysylation, carboxylation,hydroxylation, phosphorylation, and other polypeptide modificationsknown in the art.

Also, in addition to any one or more amino acid modifications, such asamino acid replacements, provided herein, modified PH20 polypeptidesprovided herein can be conjugated or fused to any moiety using anymethod known in the art, including chemical and recombinant methods,provided the resulting polypeptide retains hyaluronidase activity. Forexample, in addition to any one or more amino acid modifications, suchas amino acid replacements, provided herein, modified PH20 polypeptidesprovided herein also can contain other modifications that are or are notin the primary sequence of the polypeptide, including, but not limitedto, modification with a carbohydrate moiety, a polyethylene glycol (PEG)moiety, a sialic acid moiety, an Fc domain from immunoglobulin G, or anyother domain or moiety. For example, such additional modifications canbe made to increase the stability or serum half-life of the protein.

In some instances, the domain or other moiety is a targeted agent,including any agent that targets the conjugate to one or more cell typesby selectively binding to a cell surface receptor or other cell surfacemoiety. For example, the domain or other moiety is a targeted agent thattargets the conjugate to tumor cells. In such examples, a modified PH20polypeptide, such as any provided herein, is linked directly orindirectly to a targeted agent. Such targeting agents include, but arenot limited to, growth factors, cytokines, chemokines, antibodies, andhormones, or allelic variants, muteins, or fragments thereof so long asthe targeting agent is internalized by a cell surface receptor.Exemplary, non-limiting, additional modifications are described below.

a. Decreased Immunogenicity

The modified PH20 polypeptides provided herein can be made to havedecreased immunogenicity. Decreased immunogenicity can be effected bysequence changes that eliminate antigenic epitopes from the polypeptideor by altering post-translational modifications. One of skill in the artis familiar with methods of identifying antigenic epitopes in apolypeptide (see e.g., Liang et al. (2009) BMC Bioinformatics, 10:302;Yang et al. (2009) Rev. Med. Vivol., 19:77-96). In some examples, one ormore amino acids can be modified in order to remove or alter anantigenic epitope.

In another example, altering the glycosylation of a protein also caneffect immunogenicity. For example, altering the glycosylation of thepeptide is contemplated, so long as the polypeptides minimally containat least N-acetylglucosamine at amino acid residues corresponding toamino acid residues set forth as N200, N333 and N358 of SEQ ID NO:3 or7.

For example, the PH20 polypeptides can be modified such that they lackfucose, particularly bifucosylation. In particular, the PH20polypeptides provided herein are not bifucosylated. This can be achievedby expressing and producing the PH20 polypeptide in host cells that donot effect bifucosylation. Fucose is a deoxyhexose that is present in awide variety of organisms, including mammals, insects and plants.Fucosylated glycans are synthesized by fucosyl-transferases; see, e.g.,Ma et al., Glycobiology, 16(12):158R-184R, (2006); Nakayama et al., J.Biol. Chem., 276:16100-16106 (2001); and Sturla et al., Glycobiology,15(10):924-935 (2005). In humans, fucose frequently exists as a terminalmodification to glycan structures, and the presence of fucoseα1,6-linked to N-acetylglucosamine has been shown to be important inglycoprotein processing and recognition. In insects, N-glycan corestructures exhibit bifucosylation with α1,6- and α1,3-linkages. Insectcell core fucosylation with α1,3-linkages generates a carbohydrateepitope that is immunogenic in humans (see, e.g., US Publication No.20070067855). For example, PH20 polypeptides provided herein can begenerated in host cells that are incapable of bifucosylating thepolypeptide. Thus, while insect cells or other cells that bifucosylatecan be used for expression of the polypeptides, typically mammaliancells, such as CHO cells, are used.

In some examples, defucosylated, or fucose-deficient PH20 polypeptidescan be generated in insect cells with modified glycosylation pathways,through the use of baculovirus expression vectors containing eukaryoticoligosaccharide processing genes, thereby creating “mammalianized”insect cell expression systems (see, e.g., U.S. Pat. No. 6,461,863).Alternatively, antigenicity can be eliminated by expression of PH20polypeptides in insect cells lacking α1,3-fucosylatransferase (FT3)(see, e.g., US Publication No. 20070067855). In other examples,defucosylated or fucose-deficient PH20 polypeptides can be generated,for example, in cell lines that produce defucosylated proteins,including Lec13 CHO cells deficient in protein fucosylation (Ripka etal. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Pat. Pub. No.2003/0157108; and WO 2004/056312), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

b. Conjugation to Polymers

In some examples, the modified PH20 polypeptides provided herein areconjugated to polymers. Exemplary polymers that can be conjugated to thePH20 polypeptides, include natural and synthetic homopolymers, such aspolyols (i.e., poly-OH), polyamines (i.e., poly-NH₂) and polycarboxylicacids (i.e., poly-COOH), and further heteropolymers, i.e., polymerscontaining one or more different coupling groups, e.g., hydroxyl groupsand amine groups. Examples of suitable polymeric molecules includepolymeric molecules selected from among polyalkylene oxides (PAO), suchas polyalkylene glycols (PAG), including polyethylene glycols (PEG),methoxypolyethylene glycols (mPEG) and polypropylene glycols,PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG),branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA),polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, dextrans including carboxymethyl-dextrans, heparin,homologous albumin, celluloses, including methylcellulose,carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose,carboxyethylcellulose and hydroxypropylcellulose, hydrolysates ofchitosan, starches such as hydroxyethyl-starches andhydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guargum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acidhydrolysates and bio-polymers.

Typically, the polymers are polyalkylene oxides (PAO), such aspolyethylene oxides, such as PEG, typically mPEG, which have fewreactive groups capable of cross-linking. Typically, the polymers arenon-toxic polymeric molecules such as (methoxy)polyethylene glycol(mPEG) which can be covalently conjugated to the PH20 polypeptides(e.g., to attachment groups on the protein surface) using a relativelysimple chemistry.

Suitable polymeric molecules for attachment to the PH20 polypeptidesinclude, but are not limited to, polyethylene glycol (PEG) and PEGderivatives such as methoxy-polyethylene glycols (mPEG), PEG-glycidylethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs,and polyethylene oxide (PEO) (see e.g., Roberts et al., Advanced DrugDelivery Review 2002, 54:459-476; Harris and Zalipsky (eds.)“Poly(ethylene glycol), Chemistry and Biological Applications” ACSSymposium Series 680, 1997; Mehvar et al., J. Pharm. Pharmaceut. Sci.,3(1):125-136, 2000; Harris and Chess (2003) Nat Rev Drug Discov.2(3):214-21; and Tsubery, J Biol. Chem 279(37):38118-24, 2004). Thepolymeric molecule can be of a molecular weight typically ranging fromabout 3 kDa to about 60 kDa. In some embodiments the polymeric moleculethat is conjugated to a PH20 polypeptide provided herein has a molecularweight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60kDa.

Various methods of modifying polypeptides by covalently attaching(conjugating) a PEG or PEG derivative (i.e., “PEGylation”) are known inthe art (see e.g., U.S. 2006/0104968; U.S. Pat. Nos. 5,672,662;6,737,505; and U.S. 2004/0235734). Techniques for PEGylation include,but are not limited to, specialized linkers and coupling chemistries(see e.g., Roberts, Adv. Drug Deliv. Rev. 54:459-476, 2002), attachmentof multiple PEG moieties to a single conjugation site (such as via useof branched PEGs; see e.g., Guiotto et al., Bioorg. Med. Chem. Lett.12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation (seee.g., Chapman et al., Nature Biotech. 17:780-783, 1999), andsite-directed enzymatic PEGylation (see e.g., Sato, Adv. Drug Deliv.Rev., 54:487-504, 2002) (see, also, for example, Lu and Felix (1994)Int. J. Peptide Protein Res. 43:127-138; Lu and Felix (1993) PeptideRes. 6:140-6, 1993; Felix et al. (1995) Int. J. Peptide Res. 46:253-64;Benhar et al. (1994) J. Biol. Chem. 269:13398-404; Brumeanu et al.(1995) J Immunol. 154:3088-95; see also, Caliceti et al. (2003) Adv.Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy 23(8 Pt 2):3S-8S). Methods and techniques described in the art can produceproteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEGderivatives attached to a single protein molecule (see e.g., U.S.2006/0104968).

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, N-hydroxysuccinimidyl (NHS)activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEGsuccinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEGsuccinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acidsuccinimidyl ester, homobifunctional PEG-succinimidyl propionate,homobifunctional PEG propionaldehyde, homobifunctional PEGbutyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonatePEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEGbutryaldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEGpiperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinylsulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyldisulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylatePEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardiniet al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. BioactiveCompatible Polymers 12:197-207, 1997; U.S. Pat. Nos. 5,672,662;5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614;5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581; 5,795,569;5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237; 6,113,906;6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339; U.S. Pat. Nos.6,437,025; 6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S.2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430;U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637;US 2004/0235734; U.S. 2005/0114037; U.S. 2005/0171328; U.S.2005/0209416; EP 1064951; EP 0822199; WO 01076640; WO 0002017; WO0249673; WO 9428024; WO 0187925; and WO 2005000360).

D. METHODS FOR IDENTIFYING MODIFIED HYALURONAN-DEGRADING ENZYMES WITHALTERED PROPERTIES OR ACTIVITIES

Provided herein are methods for identifying a modified or varianthyaluronan-degrading enzyme, such as a modified hyaluronidase ormodified PH20 polypeptide, that exhibits an altered activity or propertycompared to an unmodified hyaluronan-degrading enzyme. In particular,the methods provided herein can be used to screen for one or moremodified hyaluronan-degrading enzymes, such as one or more modifiedhyaluronidase or PH20 polypeptide, that exhibits increased activityand/or increased stability in the presence of a denaturation agent orcondition. For example, the methods can be used to identify a modifiedor variant hyaluronan-degrading enzyme, such as a modified or varianthyaluronidase or modified or variant PH20 polypeptide, that exhibitsincreased stability by virtue of increased resistance to denaturationconditions, including but not limited to, denaturation conditions causedby temperature (e.g., elevated temperature such as heat), agitation, noor low salt, presence of an excipient and/or a denaturing agent.Exemplary denaturing agents or excipients include, but are not limitedto, antiadherents, binders, coatings, fillers and diluents, flavors,colors, lubricants, glidants, preservatives, sorbents or sweeteners. Forexample, various excipients, such as preservatives, can act as proteindenaturing agents. In the method, the activity also can be compared toan unmodified hyaluronan-degrading enzyme under the same denaturationcondition, and a modified hyaluronan-degrading enzyme identified orselected that exhibits greater activity than the correspondingunmodified hyaluronan-degrading enzyme.

In the method, one or more modified hyaluronan-degrading enzymes areprovided. In some examples, a library of modified molecules is prepared.Methods of mutagenesis and generation of libraries or collections ofvariant molecules is described herein and is known to one of skill inthe art using standard recombinant DNA techniques. In one example, theenzymes that are tested can be pooled and screened, whereby the methodpermits selection of only those enzymes that exhibit a desired activity.In another example, the tested enzymes can be physically separated andscreened individually, such as by formatting in arrays, such asaddressable arrays.

In one aspect of the method, the modified hyaluronan-degrading enzymesare tested or screened for hyaluronidase activity in the presence andabsence of one or more denaturation conditions or denaturing agent.After testing under both sets of conditions, the activities are assessedin order to identify modified hyaluronan-degrading enzymes that exhibitactivity in the presence of the denaturation condition. The desiredlevel or amount of activity selected as a cut-off in the methods can beempirically determined by the user, and is dependent on factors such asthe particular hyaluronan-degrading enzyme, the desired application oruse of the hyaluronan-degrading enzyme, the particular denaturationcondition or denaturing agent and other similar factors. Typically, amodified hyaluronan-degrading enzyme is identified that exhibits atleast 5% or 10% of the activity in the presence of a denaturing agent orcondition compared to in its absence, and generally at least 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, for example at least 40%of the activity.

Additionally or alternatively, the activity of the modifiedhyaluronan-degrading enzyme in the presence of one or more denaturationconditions or denaturing agents is compared to the activity of thecorresponding unmodified hyaluronan-degrading enzyme in the presence ofthe same denaturation agent(s) or condition(s). In such examples, it isunderstood that the activity of the modified and unmodified enzyme aretested under the same conditions (e.g., time, temperature, composition),except for the difference in the particular enzyme tested (unmodifiedversus modified). A modified hyaluronan-degrading enzyme is identifiedthat exhibits greater activity, such as at least 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500% or more ofthe activity of the unmodified hyaluronan-degrading enzyme.

The method can be performed a plurality of times, whereby the steps ofthe method are repeated 1, 2, 3, 4, or 5 times. The method providedherein also is iterative. In one example, after the method is performed,any identified modified hyaluronan-degrading enzyme can be modified orfurther modified to increase or optimize the activity.

A description of the steps of the method and components of the methodare provided in the subsections that follow.

1. Hyaluronan-Degrading Enzymes and Libraries of ModifiedHyaluronan-Degrading Enzymes

In the methods herein, one or more modified hyaluronan-degradingenzymes, such as a hyaluronidase or a PH20 polypeptide, are tested for adesired activity or property, such as increased stability (e.g.,increased resistance to a denaturation condition). The modifiedhyaluronan-degrading enzyme can be modified compared to an unmodifiedhyaluronan-degrading enzyme, such as any hyaluronan-degrading enzymeknown in the art. Hyaluronan-degrading enzymes are a family of enzymesthat degrade hyaluronic acid, which is an essential component of theextracellular matrix and a major constituent of the interstitialbarrier. Hyaluronan-degrading enzymes act to degrade hyaluronan bycleaving hyaluronan polymers, which are composed of repeatingdisaccharides units: D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine(GlcNAc), linked together via alternating β-1→4 and β-1→3 glycosidicbonds. By catalyzing the hydrolysis of hyaluronic acid, a majorconstituent of the interstitial barrier, hyaluronan-degrading enzymeslower the viscosity of hyaluronic acid, thereby increasing tissuepermeability. Accordingly, hyaluronan-degrading enzymes for the uses andmethods provided herein include any enzyme having the ability tocatalyze the cleavage of a hyaluronan disaccharide chain or polymer. Insome examples, the hyaluronan-degrading enzyme cleaves the β-1→4glycosidic bond in the hyaluronan chain or polymer. In other examples,the hyaluronan-degrading enzyme catalyzes the cleavage of the β-1→3glycosidic bond in the hyaluronan chain or polymer.

Hyaluronan-degrading enzymes include enzymes that are membrane-bound orthat are soluble forms that are secreted from cells. Thus, wherehyaluronan-degrading enzymes include a glycosylphosphatidylinositol(GPI) anchor signal sequence and/or are otherwise membrane-anchored orinsoluble, such hyaluronan-degrading enzymes can be provided in solubleform by C-terminal truncation or deletion of all or a portion of the GPIanchor signal sequence to render the enzyme secreted and soluble. Thus,hyaluronan-degrading enzymes include C-terminally truncated variants,e.g., truncated to remove all or a portion of a GPI anchor signalsequence. Examples of such soluble hyaluronidases are soluble PH20hyaluronides, such as any set forth in U.S. Pat. No. 7,767,429; U.S.Publication Nos. 2004/0268425 and 2010/0143457.

Exemplary hyaluronan-degrading enzymes are non-human animal or humanhyaluronidases, bacterial hyaluronidases, hyaluronidases from leeches orchondroitinases that exhibit hyaluronan-degrading activity, includingsoluble or truncated forms thereof that are active. Exemplary non-humananimal hyaluronidases are any set forth in any of SEQ ID NOs: 8-31,856-861, 869, 870, 871-886, or mature, C-terminally truncated variantsthat are soluble and active, or active forms thereof. Exemplary humanhyaluronidases are any set forth in any of SEQ ID NOs: 2, 3, 6, 7,32-66, 68-72 or 887-890, or mature, C-terminally truncated variants thatare soluble and active, or active forms thereof, and in particular anyof SEQ ID NOs: 3, 7, 32-66, 69 or 72. Exemplary bacterial hyaluronidasesare any set forth in any of SEQ ID NOs: 891-919 or mature, C-terminallytruncated variants that are soluble and active, or active forms thereof.Exemplary hyaluronidases from leeches are set forth in SEQ ID NO:920 or921, or mature, C-terminally truncated variants that are soluble andactive, or active forms thereof. Exemplary chondroitinases that havehyaluronan-degrading enzyme activity are set forth in SEQ ID NO:922-924,or mature, C-terminally truncated variants that are soluble and active,or active forms thereof.

For example, one or more modified PH20 polypeptides are tested for adesired activity or property, such as increased stability (e.g.,increased resistance to a denaturation condition). The modified PH20polypeptide can be modified compared to an unmodified PH20 polypeptide,such as any known PH20 polypeptide native, wildtype or referencepolypeptide. For example, the modified PH20 polypeptide is modifiedcompared to a full-length, soluble or active form of a PH20 polypeptide,such as any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72, or apolypeptide that exhibits at least 85%, such as at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72. In particularexamples of the method herein, the starting or unmodified PH20polypeptide has the sequence of amino acids set forth in SEQ ID NO:3.

Libraries or collections of modified hyaluronan-degrading enzymes can bescreened. Hyaluronan-degrading enzymes can be modified by any processknown to one of skill in the art that can alter the structure of aprotein. Examples of modifications include replacement, addition, anddeletion of one or more amino acids of the protein to form libraries orcollections of modified hyaluronan-degrading enzymes. It is within thelevel of one of skill in the art to generate modified or variantproteins for use in the methods herein. Methods of mutagenesis are wellknown in the art and include, for example, site-directed mutagenesissuch as for example QuikChange (Stratagene) or saturation mutagenesis.Mutagenesis methods include, but are not limited to, site-mediatedmutagenesis, PCR mutagenesis, cassette mutagenesis, site-directedmutagenesis, random point mutagenesis, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA, point mismatch repair, mutagenesis using repair-deficienthost strains, restriction-selection and restriction-purification,deletion mutagenesis, mutagenesis by total gene synthesis, double-strandbreak repair, and many others known to persons of skill. In the methodsherein, mutagenesis can be effected across the full length of a proteinor within a region of a protein. The mutations can be made rationally orrandomly.

In some examples, the methods provided herein are performed such thatthe identity of each mutant protein is known a priori before the proteinis tested. For example, the methods provided herein can be conducive tomutagenesis and screening or testing methods that are addressable. Thiscan permit the ease of comparisons between the activities of testedproteins without the need for sequencing of identified proteins. Forexample, site-directed mutagenesis methods can be used to individuallygenerate mutant proteins. Mutagenesis can be performed by thereplacement of single amino acid residues at specific target positionsone-by-one, such that each individual mutant generated is the singleproduct of each single mutagenesis reaction. Mutant DNA molecules can bedesigned, generated by mutagenesis and cloned individually, such as inaddressable arrays, such that they are physically separated from eachother and each one is the single product of an independent mutagenesisreaction. The amino acids selected to replace the target positions onthe particular protein being optimized can be either all of theremaining 19 amino acids, or a more restricted group containing onlyselected amino acids. In some methods provided herein, each amino acidthat is replaced is independently replaced by 19 of the remaining aminoacids or by less than 19 of the remaining amino acids, such as 10, 11,12, 13, 14, 15, 16, 17 or 18 of the remaining amino acids.

2. Screening or Testing For A Desired Activity or Property

The hyaluronidase activity or other activity of a composition containinga modified hyaluronan-degrading enzyme is screened or tested underconditions that expose the hyaluronan-degrading enzyme to a denaturationcondition or a denaturing agent (presence of denaturation condition ordenaturing agent). The denaturing condition or denaturing agent need notbe a condition or agent that is completely deadly to the enzyme, butgenerally is any condition or agent that destabilizes enzyme activityover time. For example, the denaturation condition can be a conditioncaused by temperature (e.g., elevated temperature such as greater thanor about or 30° C., for example, 30° C. to 42° C. such as or about 37°C.), agitation, no or low salt (e.g., NaCl), and/or caused by thepresence of a denaturing agent, such as the presence of excipients(e.g., presence of preservatives).

For purposes of selecting or identifying a modified hyaluronan-degradingenzyme that exhibits stability or increased stability under thedenaturation condition, activity can be compared to activity of themodified hyaluronan-degrading enzyme in the absence of the denaturationcondition and/or activity of the corresponding unmodifiedhyaluronan-degrading enzyme in the presence of the denaturationcondition. For example, the modified hyaluronan-degrading enzyme alsocan be screened or tested under the same conditions, except notincluding a denaturing condition or denaturing agent (absence ofdenaturation condition or denaturing agent). If desired, the activity ofthe corresponding unmodified hyaluronan-degrading enzyme (e.g., thehyaluronan-degrading enzyme not containing the amino acidreplacement(s)) can also be tested under the same conditions that exposethe hyaluronan-degrading enzyme to the same denaturation condition or adenaturing agent.

For example, each member of a library or collection of modifiedhyaluronan-degrading enzymes is incubated under or exposed to one ormore denaturation conditions. The incubation or exposure can occur invivo or in vitro. Typically, the assay is performed in vitro. The samemodified enzyme also is exposed or incubated to a reference or controlcondition that does not contain the denaturation condition. Theactivities under both conditions are compared in order to identifymodified hyaluronan-degrading enzymes that exhibit stability uponexposure to a denaturation condition or conditions. Further, inscreening or identifying the activity of the enzyme under the twodifferent sets of conditions, generally the only conditions that arevaried in the assay relate to the presence or absence of one or moredenaturation conditions. The other conditions of the assay, includingbut not limited to, time, temperature and/or other incubationconditions, can be the same for both sets of conditions.

For example, exposure can be achieved by incubation of a modifiedhyaluronan-degrading enzyme in an assay buffer or composition that hasbeen modified or adjusted to contain a denaturing agent such as anexcipient or low or no salt. Exemplary denaturing agents or excipientsinclude, but are not limited to, antiadherents, binders, coatings,fillers and diluents, flavors, colors, lubricants, glidants,preservatives, sorbents or sweeteners. The choice of buffer that is usedcan be empirically determined by one skilled in the art depending on theparticular parameter or parameters being modified. Exemplary assaybuffers are Good's buffers (see e.g., Good et al. (1966) Biochemistry,5:467-477). Examples of such buffers include, but are not limited toACES, ADA, BES, Bicine, BIS-TRIS, CAPS, HEPES, MES, MOPS, PIPES, TRIS orTrizma® buffers. Further, the amount or concentration of the excipientor salt can be empirically determined by one of skill in the artdepending on the choice of excipient or salt and the desired level oractivity of the modified hyaluronan-degrading enzyme.

In one example, the assay buffer or composition is modified by inclusionof an amount of a denaturing agent or denaturing excipient that is apreservative, for example, a phenolic preservative. The phenolicpreservative can be phenol, metacresol (m-cresol), benzyl alcohol, andparabens including methylparaben and propylparaben. In particular, thephenolic preservative is phenol and/or m-cresol. The total amount of oneor more phenolic preservative agents as a percentage (%) of massconcentration (w/v) can be between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or0.3% to 0.4% inclusive. In such an example, the activity of the modifiedhyaluronan-degrading enzyme is tested or assessed in the presence ofsuch a total amount (e.g., between or about between 0.05% to 0.6%) ofone or more preservatives, for example, one or more phenolicpreservatives. In some examples, the modified hyaluronan-degradingenzyme also can be tested or assessed under a control or referencecondition in which the assay buffer or composition is not modified tocontain a preservative. In certain instances, as a control, the activityof modified hyaluronan-degrading enzymes also can be compared to thecorresponding unmodified hyaluronan-degrading enzyme not containing themodification(s) under conditions that contain a preservative agentand/or under conditions that do not contain a preservative agent.

In another example, the assay buffer is modified by the presence of adenaturation condition that is low or no salt. As discussed elsewhereherein, hyaluronan-degrading enzymes, such as PH20, generally requiresalt (e.g., NaCl, Lys-Lys or MgCl₂) for activity. Hence, the absence ofsalt or low salt is denaturing to the enzyme. In one example, the assaybuffer is modified by inclusion of an amount of salt that is less than100 mM, for example, less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM,30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. In such an example, theactivity of the modified hyaluronan-degrading enzyme is tested in theabsence of salt or in the presence of salt that is less than 100 mM. Insome examples, the modified hyaluronan-degrading enzyme also can betested or assessed under a control or reference condition in which theassay buffer contains a higher salt concentration, generally between orabout between 140 mM to 200 mM. In certain instances, as a control, theactivity of modified hyaluronan-degrading enzymes also can be comparedto the corresponding unmodified hyaluronan-degrading enzyme notcontaining the modification(s) under conditions that contain low or nosalt, such as less than 100 mM and/or under conditions that contain saltin an amount that is between or about between 140 mM to 200 mM.

Exposure of a hyaluronan-degrading enzyme to a denaturation conditionalso can be achieved by incubation of a modified hyaluronan-degradingenzyme under conditions that are known to be denaturing, such as underconditions of elevated temperature such as a temperature greater than orabout or 30° C. (e.g., 30° C. to 42° C. such as or about 37° C.) oragitation. For example, the activity of the modifiedhyaluronan-degrading enzyme is tested at elevated temperatures greaterthan or about or 30° C. to 42° C. In some examples, the modifiedhyaluronan-degrading enzyme also can be tested or assessed under acontrol or reference condition where the temperatures is less than 30°C., such as between or about between 0° C. to 25° C., for example, 0° C.to 5° C. or 18° C. to 25° C. In certain instances, as a control, theactivity of modified hyaluronan-degrading enzymes also can be comparedto the corresponding unmodified hyaluronan-degrading enzyme notcontaining the modification(s) under elevated temperatures greater thanor about or 30° C. to 42° C. and/or temperatures is less than 30° C.,such as between or about between 0° C. to 25° C., for example, 0° C. to5° C. or 18° C. to 25° C.

The modified hyaluronan-degrading enzyme can be exposed to one or morethan one of the conditions. The exposure to one condition can occursimultaneously, subsequently, intermittently or periodically to exposureto one or more other conditions.

In one example, in the method herein, the modified hyaluronan-degradingenzyme is incubated or exposed to the denaturation condition ordenaturing agent prior to performing an assay for hyaluronidaseactivity. For example, the modified hyaluronan-degrading enzyme isincubated in the presence of a denaturing agent or exposed to one ormore denaturation conditions or control conditions, such as one or moreof the denaturation conditions or control conditions as described above.The incubation or exposure can be for any desired length of time, andcan be empirically determined by one of skill in the art. For example,the modified hyaluronan-degrading enzyme can be incubated or exposed toone or more denaturation conditions, denaturing agents or controlconditions for or about for 1 minute to 1 month, such as 1 minute to 3weeks, 1 minute to 2 weeks, 1 minute to 1 week, 1 minute to 24 hours, 1minute to 12 hours, such as 30 minutes to 6 hours or 1 hour to 4 hours,and generally at least or about at least 30 minutes, 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,11 hours or 12 hours. After the time of incubation or exposure, thesample or composition containing the modified hyaluronan-degradingenzyme (or control unmodified enzyme) is assessed for hyaluronidaseassay. In another example, the modified hyaluronan-degrading enzyme isexposed or incubated under one or more denaturation conditions and issimultaneously or concurrently assessed for hyaluronidase activity. Inany examples where a modified hyaluronan-degrading enzyme is assessed,it is understood that an unmodified hyaluronan-degrading enzyme notcontaining the modifications(s) also can be assessed under similar assayconditions for comparison.

Assays to assess hyaluronidase activity are well known in the art.Examples of such assays are described in Section G. In one example,hyaluronidase activity can be assessed in a microturbidity assay,wherein the amount of undegraded HA is measured by the addition of areagent that precipitates HA (e.g., Cetylpyridinium chloride (CPC) oracidified serum) after the enzyme is allowed to react with HA. Inanother example, hyaluronidase activity can be assessed using amicrotiter assay in which residual biotinylated hyaluronic acid ismeasured following incubation with hyaluronidase (see e.g., Frost andStern (1997) Anal. Biochem. 251:263-269, U.S. Pat. Publication No.20050260186). The resulting activities under each of the testedconditions is determined and compared.

3. Selection or Identification

In the method, after screening modified hyaluronan-degrading enzymesunder one or more denaturation conditions, the hyaluronidase activitiesof the tested enzymes are compared. The method is practiced in order toidentify a modified hyaluronan-degrading enzyme that is more resistantto denaturation by a condition or a denaturing agent, whereby theactivity of the enzyme is indicative of the stability of the enzyme as ameasure of its resistance to denaturation. It is understood that somereduction of enzyme activity, as a result of denaturation, can betolerated in various applications, and thus the method can be practicedto select for a modified hyaluronan-degrading enzymes that exhibits arequisite activity upon exposure to a denaturation condition to permitits use or application (e.g., therapeutic activity). For example, amodified enzyme can be selected that loses activity more slowly than thecorresponding unmodified or reference hyaluronan-degrading enzyme, butwhose retained activity is sufficient for a particular application orpurpose.

In examples of the methods herein, the activity of the modifiedhyaluronan degrading enzyme is assessed upon exposure to a firstdenaturation condition and also assessed upon exposure to a secondcondition that is a control or non-denaturation condition, and theresulting hyaluronidase activities compared. For comparison, in someexamples, the activity can be represented as a ratio of activity or apercentage of activity under a denaturation condition compared to undera control or non-denaturation condition. For example, where theparameter that differs between the first and second condition is thepresence of preservative (e.g., phenolic preservative), activity can berepresented as a ratio of activity or percentage of activity observed inthe presence of preservative (e.g., phenolic preservative) versusactivity in the absence of preservative (e.g., phenolic preservative).In another example, where the parameter that differs between the firstand second condition is temperature, activity can be represented as aratio of activity or percentage of activity observed in the presence ofelevated temperature (e.g., 30° C. to 42° C.) compared to activity inthe presence of a lower temperature such as 0° C. to 25° C., for example0° C. to 5° C. or 18° C. to 25° C.

A modified hyaluronan-degrading enzyme is selected or identified thatretains or exhibits any desired activity in the presence of thedenaturation condition compared to in its absence. The particularcut-off of activity for selection of enzymes herein is dependent on theparticular user and/or practice of the method and can be empiricallydetermined depending on factors such as the particular denaturationcondition or denaturing agent, the particular modifiedhyaluronan-degrading enzyme, the desired application of the identifiedor selected hyaluronan-degrading enzyme and other similar factors.Generally, a selected or identified modified hyaluronan-degrading enzymeexhibits stability if any detectable activity is measured or assessedupon exposure or incubation with a denaturation condition or denaturingagent. For example, a selected or identified modifiedhyaluronan-degrading enzyme exhibits stability, or resistance to adenaturation condition or denaturing agent, if it exhibits at least 5%or 10% of the activity of the same enzyme in the absence of thedenaturation condition or denaturing agent, and generally if themodified hyaluronan-degrading enzyme exhibits an activity that is atleast 15% of the initial hyaluronidase activity prior to incubation inthe presence of the denaturation condition. For example, a modifiedhyaluronan-degrading enzyme is selected or identified that exhibits atleast (or at least about) 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%,130%, 140%, 150%, 200%, 300%, 400%, 500% or more of the initialhyaluronidase activity of the modified hyaluronan-degrading enzymetested under a control or non-denaturation condition.

In other examples of the methods herein, the activity of the modifiedhyaluronan degrading enzyme is assessed upon exposure to a denaturationcondition and the activity of the unmodified or referencehyaluronan-degrading enzyme also is assessed upon exposure to the samedenaturation conditions. In such examples, the activities are comparedwhen the enzymes are exposed to the same conditions. For comparison, theactivity under a denaturation condition can be represented as a ratio ofactivity or a percentage of activity of a modified hyaluronan-degradingenzyme compared to an unmodified or reference hyaluronan-degradingenzyme. In such examples, a modified hyaluronan-degrading enzyme isselected that exhibits greater activity under a denaturation conditionthan the unmodified or reference hyaluronan-degrading enzyme. Thus, themodified hyaluronan-degrading enzyme is one that is more resistant tothe condition. For example, where the denaturation condition is thepresence of preservative (e.g., phenolic preservative), the activityobserved in the presence of preservative (e.g., phenolic preservative)can be represented as a ratio of activity or percentage of activity ofthe modified hyaluronan-degrading enzyme compared to the unmodified orreference hyaluronan-degrading enzyme. In another example, where thedenaturation condition is high temperature, activity observed in thepresence of elevated temperature (e.g., 30° C. to 42° C.) can berepresented as a ratio of activity or percentage of activity of themodified hyaluronan-degrading enzyme compared to the unmodified orreference hyaluronan-degrading enzyme.

In such examples, a modified hyaluronan-degrading enzyme, such as amodified PH20, is identified or selected that exhibits a ratio ofactivity that is greater than or at least 1.1, such that the enzymeexhibits greater activity than the unmodified or referencehyaluronan-degrading enzyme under the denaturation condition. Forexample, the ratio is at least or at least about 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 orgreater. A modified hyaluronan-degrading enzyme (e.g., a modified PH20)can be selected if its activity is at least 120%, 130%, 140%, 150%,160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500% or more of theactivity of the unmodified or reference hyaluronan-degrading enzyme whentested under the same conditions. Thus, modified hyaluronan-degradingenzymes are identified that exhibit greater or improved stabilitycompared to the unmodified hyaluronan-degrading enzyme or a referencehyaluronan-degrading enzyme as manifested by increased resistance to adenaturation condition or denaturing agent.

4. Iterative Methods

The method provided herein also is iterative. In one example, after themethod is performed, any modified hyaluronan-degrading enzymesidentified as exhibiting stability, such as increased stability, under adenaturation condition can be modified or further modified to increaseor optimize the stability. A secondary library can be created byintroducing additional modifications in a first identified modifiedhyaluronan-degrading enzyme. For example, modifications that wereidentified as conferring stability, such as increasing stability, can becombined to generate a combinatorial library. The secondary library canbe tested using the assays and methods described herein.

In another example of an iterative aspect of the method, modifiedhyaluronan-degrading enzymes that are identified as not exhibitingstability such as increased stability (e.g., such that they are notactive or do not have increased activity under the a denaturationcondition), can be further modified and retested for stability under adenaturation condition. The further modifications can be targeted nearparticular regions (e.g., particular amino acid residues) associatedwith activity and/or stability of the molecule. For example, residuesthat are associated with activity and/or stability of the moleculegenerally are critical residues that are involved in the structuralfolding or other activities of the molecule. Hence, such residues arerequired for activity, generally under any condition. Critical residuescan be identified because, when mutated, a normal activity of theprotein is ablated or reduced. For example, critical residues can beidentified that, when mutated in a hyaluronan-degrading enzyme, exhibitreduced or ablated hyaluronidase activity under a normal or controlassay condition. A further library of modified proteins can be generatedwith amino acid mutations targeted at or near to the identified criticalamino acid residues, such as adjacent to the identified critical aminoacid residues. In some examples, the mutations can be amino acidreplacement to any other of up to 19 other amino acid residues. Thesecondary library can be tested using the assays and methods describedherein.

E. PRODUCTION OF MODIFIED PH20 POLYPEPTIDES AND ENCODING NUCLEIC ACIDMOLECULES

Polypeptides of a modified PH20 polypeptide set forth herein can beobtained by methods well known in the art for protein purification andrecombinant protein expression. Polypeptides also can be synthesizedchemically. Modified or variant, including truncated, forms can beengineered from a wildtype polypeptide using standard recombinant DNAmethods. For example, modified PH20 polypeptides can be engineered froma wildtype polypeptide, such as by site-directed mutagenesis.

1. Isolation or Preparation of Nucleic Acids Encoding PH20 Polypeptides

Polypeptides can be cloned or isolated using any available methods knownin the art for cloning and isolating nucleic acid molecules. Suchmethods include PCR amplification of nucleic acids and screening oflibraries, including nucleic acid hybridization screening,antibody-based screening and activity-based screening. For example, whenthe polypeptides are produced by recombinant means, any method known tothose of skill in the art for identification of nucleic acids thatencode desired genes can be used. Any method available in the art can beused to obtain a full length or partial (i.e., encompassing the entirecoding region) cDNA or genomic DNA clone encoding a PH20, such as from acell or tissue source.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a desired polypeptide, including forexample, polymerase chain reaction (PCR) methods. Examples of suchmethods include use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp). A nucleic acid containing material can be used asa starting material from which a desired polypeptide-encoding nucleicacid molecule can be isolated. For example, DNA and mRNA preparations,cell extracts, tissue extracts, fluid samples (e.g., blood, serum,saliva), samples from healthy and/or diseased subjects can be used inamplification methods. The source can be from any eukaryotic speciesincluding, but not limited to, vertebrate, mammalian, human, porcine,bovine, feline, avian, equine, canine, and other primate sources.Nucleic acid libraries also can be used as a source of startingmaterial. Primers can be designed to amplify a desired polypeptide. Forexample, primers can be designed based on expressed sequences from whicha desired polypeptide is generated. Primers can be designed based onback-translation of a polypeptide amino acid sequence. If desired,degenerate primers can be used for amplification. Oligonucleotideprimers that hybridize to sequences at the 3′ and 5′ termini of thedesired sequence can be uses as primers to amplify by PCR sequences froma nucleic acid sample. Primers can be used to amplify the entirefull-length PH20, or a truncated sequence thereof, such as a nucleicacid encoding any of the soluble PH20 polypeptides provided herein.Nucleic acid molecules generated by amplification can be sequenced andconfirmed to encode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art. For example, exemplary heterologoussignal sequences include, but are not limited to, human and mouse kappaIgG heterologous signal sequences set forth in SEQ ID NO: 868.Additional nucleotide residue sequences such as sequences of basesspecifying protein binding regions also can be linked to enzyme-encodingnucleic acid molecules. Such regions include, but are not limited to,sequences of residues that facilitate or encode proteins that facilitateuptake of an enzyme into specific target cells, or otherwise alterpharmacokinetics of a product of a synthetic gene.

In addition, tags or other moieties can be added, for example, to aid indetection or affinity purification of the polypeptide. For example,additional nucleotide residue sequences such as sequences of basesspecifying an epitope tag or other detectable marker also can be linkedto enzyme-encoding nucleic acid molecules. Examples of such sequencesinclude nucleic acid sequences encoding a His tag or Flag Tag.

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). Other expression vectors include theHZ24 expression vector exemplified herein (see e.g., SEQ ID NOs:4 and5). The insertion into a cloning vector can, for example, beaccomplished by ligating the DNA fragment into a cloning vector whichhas complementary cohesive termini. Insertion can be effected using TOPOcloning vectors (Invitrogen, Carlsbad, Calif.).

If the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules can beenzymatically modified. Alternatively, any site desired can be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers can contain specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and protein genecan be modified by homopolymeric tailing.

Recombinant molecules can be introduced into host cells via, forexample, transformation, transfection, infection, electroporation andsonoporation, so that many copies of the gene sequence are generated. Inspecific embodiments, transformation of host cells with recombinant DNAmolecules that incorporate the isolated protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

In addition to recombinant production, modified PH20 polypeptidesprovided herein can be produced by direct peptide synthesis usingsolid-phase techniques (see e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, WH Freeman Co., San Francisco; Merrifield J (1963) JAm Chem Soc., 85:2149-2154). In vitro protein synthesis can be performedusing manual techniques or by automation. Automated synthesis can beachieved, for example, using Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer, Foster City Calif.) in accordance with the instructionsprovided by the manufacturer. Various fragments of a polypeptide can bechemically synthesized separately and combined using chemical methods.

2. Generation of Mutant or Modified Nucleic Acid and EncodingPolypeptides

The modifications provided herein can be made by standard recombinantDNA techniques such as are routine to one of skill in the art. Anymethod known in the art to effect mutation of any one or more aminoacids in a target protein can be employed. Methods include standardsite-directed mutagenesis (using e.g., a kit, such as QuikChangeavailable from Stratagene) of encoding nucleic acid molecules, or bysolid phase polypeptide synthesis methods.

3. Vectors and Cells

For recombinant expression of one or more of the desired proteins, suchas any modified PH20 polypeptide described herein, the nucleic acidcontaining all or a portion of the nucleotide sequence encoding theprotein can be inserted into an appropriate expression vector, i.e., avector that contains the necessary elements for the transcription andtranslation of the inserted protein coding sequence. The necessarytranscriptional and translational signals also can be supplied by thenative promoter for enzyme genes, and/or their flanking regions.

Also provided are vectors that contain a nucleic acid encoding theenzyme. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein. Generally, the cell is a cell that is capableof effecting glyosylation of the encoded protein.

Prokaryotic and eukaryotic cells containing the vectors are provided.Such cells include bacterial cells, yeast cells, fungal cells, Archea,plant cells, insect cells and animal cells. The cells are used toproduce a protein thereof by growing the above-described cells underconditions whereby the encoded protein is expressed by the cell, andrecovering the expressed protein. For purposes herein, for example, theenzyme can be secreted into the medium.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the polypeptide include,but are not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingcan impact the folding and/or function of the polypeptide. Differenthost cells, such as, but not limited to, CHO (DG44, DXB11, CHO-K1),HeLa, MCDK, 293 and W138 have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and canbe chosen to ensure the correct modification and processing of theintroduced protein. Generally, the choice of cell is one that is capableof introducing N-linked glycosylation into the expressed polypeptide.Hence, eukaryotic cells containing the vectors are provided.

Exemplary eukaryotic cells are mammalian Chinese Hamster Ovary (CHO)cells. For example, CHO cells deficient in dihydrofolate reductase(e.g., DG44 cells) are used to produce polypeptides provided herein.Note that bacterial expression of an PH20 polypeptide provided hereinwill not result in a catalytically active polypeptide, but when combinedwith proper glycosylation machinery, the PH20 can be artificiallyglycosylated.

Provided are vectors that contain a sequence of nucleotides that encodesthe modified PH20 polypeptide, coupled to the native or heterologoussignal sequence, as well as multiple copies thereof. The vectors can beselected for expression of the enzyme protein in the cell or such thatthe enzyme protein is expressed as a secreted protein.

A variety of host-vector systems can be used to express the proteinencoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include, but are not limited to, the SV40 earlypromoter (Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vector promoters, suchas the β-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci.USA 78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci.USA 80:21-25 (1983); see also Gilbert and Villa-Komaroff, “UsefulProteins from Recombinant Bacteria,” Scientific American 242:74-94(1980)); plant expression vector promoters, such as the nopalinesynthetase promoter (Herrera-Estrella et al., Nature 303:209-213 (1984))or the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,Nucleic Acids Res. 9:2871 (1981)), and the promoter of thephotosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:425-515 (1987)); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., Nature 315:115-122(1985)), immunoglobulin gene control region which is active in lymphoidcells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-495 (1986)), albumin gene control region which is active in liver(Pinkert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al., Genes and Devel. 1:161-171 (1987)), beta globin gene controlregion which is active in myeloid cells (Magram et al., Nature315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., Cell 48:703-712 (1987)), myosin lightchain-2 gene control region which is active in skeletal muscle (Shani,Nature 314:283-286 (1985)), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a desired protein, or adomain, fragment, derivative or homolog thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Depending on the expression system,specific initiation signals also are required for efficient translationof a PH20 sequence. These signals include the ATG initiation codon andadjacent sequences. In cases where the initiation codon and upstreamsequences of PH20 or soluble forms thereof are inserted into theappropriate expression vector, no additional translational controlsignals are needed. In cases where only a coding sequence, or a portionthereof, is inserted, exogenous transcriptional control signalsincluding the ATG initiation codon must be provided. Furthermore, theinitiation codon must be in the correct reading frame to ensuretranscription of the entire insert. Exogenous transcriptional elementsand initiation codons can be of various origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof enhancers appropriate to the cell system in use (Scharf et al. (1994)Results Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods inEnzymol, 153:516-544).

Exemplary plasmid vectors for transformation of E. coli cells include,for example, the pQE expression vectors (available from Qiagen,Valencia, Calif.; see also literature published by Qiagen describing thesystem). pQE vectors have a phage T5 promoter (recognized by E. coli RNApolymerase) and a double lac operator repression module to providetightly regulated, high-level expression of recombinant proteins in E.coli, a synthetic ribosomal binding site (RBS II) for efficienttranslation, a 6×His tag coding sequence, to and T1 transcriptionalterminators, ColE1 origin of replication, and a beta-lactamase gene forconferring ampicillin resistance. The pQE vectors enable placement of a6×His tag at either the N- or C-terminus of the recombinant protein.Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiplecloning sites for all three reading frames and provide for theexpression of N-terminally 6×His-tagged proteins. Other exemplaryplasmid vectors for transformation of E. coli cells, include, forexample, the pET expression vectors (see, U.S. Pat. No. 4,952,496;available from Novagen, Madison, Wis.; see, also literature published byNovagen describing the system). Such plasmids include pET 11a, whichcontains the T7lac promoter, T7 terminator, the inducible E. coli lacoperator, and the lac repressor gene; pET 12a-c, which contains the T7promoter, T7 terminator, and the E. coli ompT secretion signal; and pET15b and pET19b (Novagen, Madison, Wis.), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

Typically, vectors can be plasmids, viral vectors, or others known inthe art, used for expression of the modified PH20 polypeptide in vivo orin vitro. For example, the modified PH20 polypeptide is expressed inmammalian cells, including, for example, Chinese Hamster Ovary (CHO)cells. An exemplary vector for mammalian cell expression is the HZ24expression vector. The HZ24 expression vector was derived from the pCIvector backbone (Promega). It contains DNA encoding the Beta-lactamaseresistance gene (AmpR), an F1 origin of replication, a Cytomegalovirusimmediate-early enhancer/promoter region (CMV), and an SV40 latepolyadenylation signal (SV40). The expression vector also has aninternal ribosome entry site (IRES) from the ECMV virus (Clontech) andthe mouse dihydrofolate reductase (DHFR) gene.

Viral vectors, such as adenovirus, retrovirus or vaccinia virus vectors,can be employed. In some examples, the vector is a defective orattenuated retroviral or other viral vector (see U.S. Pat. No.4,980,286). For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA.

In some examples, viruses armed with a nucleic acid encoding a modifiedPH20 polypeptide can facilitate their replication and spread within atarget tissue for example. The target tissue can be a cancerous tissuewhereby the virus is capable of selective replication within the tumor.The virus can also be a non-lytic virus wherein the virus selectivelyreplicates under a tissue specific promoter. As the viruses replicate,the coexpression of the PH20 polypeptide with viral genes willfacilitate the spread of the virus in vivo.

4. Expression

Modified PH20 polypeptides can be produced by any method known to thoseof skill in the art including in vivo and in vitro methods. Desiredproteins can be expressed in any organism suitable to produce therequired amounts and forms of the proteins, such as for example, thoseneeded for administration and treatment. Expression hosts includeprokaryotic and eukaryotic organisms such as E. coli, yeast, plants,insect cells, mammalian cells, including human cell lines and transgenicanimals. Expression hosts can differ in their protein production levelsas well as the types of post-translational modifications that arepresent on the expressed proteins. The choice of expression host can bemade based on these and other factors, such as regulatory and safetyconsiderations, production costs and the need and methods forpurification.

Many expression vectors are available and known to those of skill in theart and can be used for expression of proteins. The choice of expressionvector will be influenced by the choice of host expression system. Ingeneral, expression vectors can include transcriptional promoters andoptionally enhancers, translational signals, and transcriptional andtranslational termination signals. Expression vectors that are used forstable transformation typically have a selectable marker which allowsselection and maintenance of the transformed cells. In some cases, anorigin of replication can be used to amplify the copy number of thevector.

Modified PH20 polypeptides also can be utilized or expressed as proteinfusions. For example, an enzyme fusion can be generated to addadditional functionality to an enzyme. Examples of enzyme fusionproteins include, but are not limited to, fusions of a signal sequence,a tag such as for localization, e.g., a 6×His or His₆ tag or a myc tag,or a tag for purification, for example, a GST fusion, and a sequence fordirecting protein secretion and/or membrane association.

For long-term, high-yield production of recombinant proteins, stableexpression is desired. For example, cell lines that stably express amodified PH20 polypeptide can be transformed using expression vectorsthat contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells can be allowed to grow for 1-2 days in an enriched mediumbefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells that successfully express theintroduced sequences. Resistant cells of stably transformed cells can beproliferated using tissue culture techniques appropriate to the celltypes.

Any number of selection systems can be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M et al. (1977) Cell, 11:223-32) and adeninephosphoribosyltransferase (Lowy, I et al. (1980) Cell, 22:817-23) genes,which can be employed in TK- or APRT-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection. For example, DHFR, which confers resistance tomethotrexate (Wigler, M et al. (1980) Proc. Natl. Acad. Sci,77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F et al. (1981) J. Mol. Biol.,150:1-14); and also or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively, can be used. Additionalselectable genes have been described, for example, trpB, which allowscells to utilize indole in place of typtophan or hisD, which allowscells to utilize histinol in place of histidine (Hartman S C and RCMulligan (1988) Proc. Natl. Acad. Sci, 85:8047-51). Visible markers,such as but not limited to, anthocyanins, beta glucuronidase and itssubstrate, GUS, and luciferase and its substrate luciferin, also can beused to identify transformants and also to quantify the amount oftransient or stable protein expression attributable to a particularvector system (Rhodes C A et al. (1995) Methods Mol. Biol. 55:121-131).

The presence and expression of PH20 polypeptides can be monitored. Forexample, detection of a functional polypeptide can be determined bytesting the conditioned media for hyaluronidase enzyme activity underappropriate conditions. Exemplary assays to assess the solubility andactivity of expressed proteins are provided herein.

a. Prokaryotic Cells

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins. Transformation of E. coli is a simple and rapidtechnique well known to those of skill in the art. Expression vectorsfor E. coli can contain inducible promoters. Such promoters are usefulfor inducing high levels of protein expression and for expressingproteins that exhibit some toxicity to the host cells. Examples ofinducible promoters include the lac promoter, the trp promoter, thehybrid tac promoter, the T7 and SP6 RNA promoters and the temperatureregulated λPL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. The cytoplasm is a reducingenvironment, and for some molecules, this can result in the formation ofinsoluble inclusion bodies. Reducing agents such as dithiothreotol andβ-mercaptoethanol and denaturants, such as guanidine-HCl and urea can beused to resolubilize the proteins. An alternative approach effectsprotein expression in the periplasmic space of bacteria which providesan oxidizing environment and chaperonin-like and disulfide isomerases,which can aid in the production of soluble protein. Typically, a leadersequence is fused to the protein to be expressed which directs theprotein to the periplasm. The leader is then removed by signalpeptidases inside the periplasm. Examples of periplasmic-targetingleader sequences include the pelB leader from the pectate lyase gene andthe leader derived from the alkaline phosphatase gene. In some cases,periplasmic expression allows leakage of the expressed protein into theculture medium. The secretion of proteins allows quick and simplepurification from the culture supernatant. Proteins that are notsecreted can be obtained from the periplasm by osmotic lysis. Similar tocytoplasmic expression, in some cases proteins can become insoluble anddenaturants and reducing agents can be used to facilitate solubilizationand refolding. Temperature of induction and growth also can influenceexpression levels and solubility, typically temperatures between 25° C.and 37° C. are used. Typically, bacteria produce aglycosylated proteins.Thus, if proteins require glycosylation for function, glycosylation canbe added in vitro after purification from host cells.

b. Yeast Cells

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used for production ofproteins, such as any described herein. Yeast can be transformed withepisomal replicating vectors or by stable chromosomal integration byhomologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GALTand GALS and metallothionein promoters, such as CUP1, AOX1 or otherPichia or other yeast promoters. Expression vectors often include aselectable marker such as LEU2, TRP1, HIS3 and URA3 for selection andmaintenance of the transformed DNA. Proteins expressed in yeast areoften soluble. Co-expression with chaperonins such as Bip and proteindisulfide isomerase can improve expression levels and solubility.Additionally, proteins expressed in yeast can be directed for secretionusing secretion signal peptide fusions such as the yeast mating typealpha-factor secretion signal from Saccharomyces cerevisae and fusionswith yeast cell surface proteins such as the Aga2p mating adhesionreceptor or the Arxula adeninivorans glucoamylase. A protease cleavagesite such as for the Kex-2 protease, can be engineered to remove thefused sequences from the expressed polypeptides as they exit thesecretion pathway. Yeast also is capable of glycosylation atAsn-X-Ser/Thr motifs.

c. Insects and Insect Cells

Insect cells, particularly using baculovirus expression, are useful forexpressing polypeptides such as PH20 polypeptides. Insect cells expresshigh levels of protein and are capable of most of the post-translationalmodifications used by higher eukaryotes. Baculoviruses have arestrictive host range which improves the safety and reduces regulatoryconcerns of eukaryotic expression. Typical expression vectors use apromoter for high level expression such as the polyhedrin promoter ofbaculovirus. Commonly used baculovirus systems include a baculovirus,such as the Autographa californica nuclear polyhedrosis virus (AcNPV) orthe Bombyx mori nuclear polyhedrosis virus (BmNPV), and an insect cellline, such as Sf9 derived from Spodoptera frugiperda, Pseudaletiaunipuncta (A7S) and Danaus plexippus (DpN1). For high-level expression,the nucleotide sequence of the molecule to be expressed is fusedimmediately downstream of the polyhedrin initiation codon of the virus.Mammalian secretion signals are accurately processed in insect cells andcan be used to secrete the expressed protein into the culture medium. Inaddition, the cell lines Pseudaletia unipuncta (A7S) and Danausplexippus (DpN1) produce proteins with glycosylation patterns similar tomammalian cell systems. Exemplary insect cells are those that have beenaltered to reduce immunogenicity, including those with “mammalianized”baculovirus expression vectors and those lacking the enzyme FT3.

An alternative expression system in insect cells employs stablytransformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Expression

Mammalian expression systems can be used to express proteins includingPH20 polypeptides. Expression constructs can be transferred to mammaliancells by viral infection such as by adenovirus or by direct DNA transfersuch as liposomes, calcium phosphate, DEAE-dextran and by physical meanssuch as electroporation and microinjection. Expression vectors formammalian cells typically include an mRNA cap site, a TATA box, atranslational initiation sequence (Kozak consensus sequence) andpolyadenylation elements. IRES elements also can be added to permitbicistronic expression with another gene, such as a selectable marker.Such vectors often include transcriptional promoter-enhancers forhigh-level expression, for example the SV40 promoter-enhancer, the humancytomegalovirus (CMV) promoter and the long terminal repeat of Roussarcoma virus (RSV). These promoter-enhancers are active in many celltypes. Tissue and cell-type promoters and enhancer regions also can beused for expression. Exemplary promoter/enhancer regions include, butare not limited to, those from genes such as elastase I, insulin,immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein,alpha 1 antitrypsin, beta globin, myelin basic protein, myosin lightchain 2, and gonadotropic releasing hormone gene control. Selectablemarkers can be used to select for and maintain cells with the expressionconstruct. Examples of selectable marker genes include, but are notlimited to, hygromycin B phosphotransferase, adenosine deaminase,xanthine-guanine phosphoribosyl transferase, aminoglycosidephosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase.For example, expression can be performed in the presence of methotrexateto select for only those cells expressing the DHFR gene. Fusion withcell surface signaling molecules such as TCR-ζ and Fc_(ε)RI-γ can directexpression of the proteins in an active state on the cell surface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, chicken and hamster cells. Exemplary cell linesinclude but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NS0(nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are availableadapted to serum-free media which facilitates purification of secretedproteins from the cell culture media. Examples include CHO—S cells(Invitrogen, Carlsbad, Calif., cat #11619-012) and the serum free EBNA-1cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42). Celllines also are available that are adapted to grow in special mediumsoptimized for maximal expression. For example, DG44 CHO cells areadapted to grow in suspension culture in a chemically defined, animalproduct-free medium.

e. Plants and Plant Cells

Transgenic plant cells and plants can be used to express proteins suchas any described herein. Expression constructs are typically transferredto plants using direct DNA transfer such as microprojectile bombardmentand PEG-mediated transfer into protoplasts, and withagrobacterium-mediated transformation. Expression vectors can includepromoter and enhancer sequences, transcriptional termination elementsand translational control elements. Expression vectors andtransformation techniques are usually divided between dicot hosts, suchas Arabidopsis and tobacco, and monocot hosts, such as corn and rice.Examples of plant promoters used for expression include the cauliflowermosaic virus promoter, the nopaline synthase promoter, the ribosebisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.Selectable markers such as hygromycin, phosphomannose isomerase andneomycin phosphotransferase are often used to facilitate selection andmaintenance of transformed cells. Transformed plant cells can bemaintained in culture as cells, aggregates (callus tissue) orregenerated into whole plants. Transgenic plant cells also can includealgae engineered to produce hyaluronidase polypeptides. Because plantshave different glycosylation patterns than mammalian cells, this caninfluence the choice of protein produced in these hosts.

5. Purification

Host cells transformed with a nucleic acid sequence encoding a modifiedPH20 polypeptide can be cultured under conditions suitable for theexpression and recovery of the encoded protein from cell culture. Theprotein produced by a recombinant cell is generally secreted, but may becontained intracellularly depending on the sequence and/or the vectorused. As will be understood by those of skill in the art, expressionvectors containing nucleic acid encoding PH20 can be designed withsignal sequences that facilitate direct secretion of PH20 throughprokaryotic or eukaryotic cell membranes.

Thus, methods for purification of polypeptides from host cells willdepend on the chosen host cells and expression systems. For secretedmolecules, proteins are generally purified from the culture media afterremoving the cells. For intracellular expression, cells can be lysed andthe proteins purified from the extract. When transgenic organisms suchas transgenic plants and animals are used for expression, tissues ororgans can be used as starting material to make a lysed cell extract.Additionally, transgenic animal production can include the production ofpolypeptides in milk or eggs, which can be collected, and if necessary,the proteins can be extracted and further purified using standardmethods in the art.

Proteins, such as modified PH20 polypeptides, can be purified usingstandard protein purification techniques known in the art including butnot limited to, SDS-PAGE, size fractionation and size exclusionchromatography, ammonium sulfate precipitation and ionic exchangechromatography, such as anion exchange chromatography. Affinitypurification techniques also can be utilized to improve the efficiencyand purity of the preparations. For example, antibodies, receptors andother molecules that bind PH20 hyaluronidase enzymes can be used inaffinity purification. For example, soluble PH20 can be purified fromconditioned media.

Expression constructs also can be engineered to add an affinity tag to aprotein such as a myc epitope, GST fusion or His₆ and affinity purifiedwith myc antibody, glutathione resin or Ni-resin, respectively. Suchtags can be joined to the nucleotide sequence encoding a soluble PH20 asdescribed elsewhere herein, which can facilitate purification of solubleproteins. For example, a modified PH20 polypeptide can be expressed as arecombinant protein with one or more additional polypeptide domainsadded to facilitate protein purification. Such purification facilitatingdomains include, but are not limited to, metal chelating peptides suchas histidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego, Calif.) between the purification domain and the expressedPH20 polypeptide is useful to facilitate purification. One suchexpression vector provides for expression of a fusion protein containinga PH20 polypeptide in and an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography), while the enterokinase cleavage site providesa means for purifying the polypeptide from the fusion protein.

Purity can be assessed by any method known in the art including gelelectrophoresis, orthogonal HPLC methods, staining andspectrophotometric techniques. The expressed and purified protein can beanalyzed using any assay or method known to one of skill in the art, forexample, any described in Section G. These include assays based on thephysical and/or functional properties of the protein, including, but notlimited to, analysis by gel electrophoresis, immunoassay and assays ofhyaluronidase activity.

Depending on the expression system and host cells used, the resultingpolypeptide can be heterogeneous due to peptidases present in theculture medium upon production and purification. For example, culture ofsoluble PH20 in CHO cells can result in a mixture of heterogeneouspolypeptides.

6. Modification of Polypeptides by PEGylation

Polyethylene glycol (PEG) has been widely used in biomaterials,biotechnology and medicine primarily because PEG is a biocompatible,nontoxic, water-soluble polymer that is typically nonimmunogenic (Zhaoand Harris, ACS Symposium Series 680: 458-72, 1997). In the area of drugdelivery, PEG derivatives have been widely used in covalent attachment(i.e., “PEGylation”) to proteins to reduce immunogenicity, proteolysisand kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del.Rev. 16:157-82, 1995). Similarly, PEG has been attached to low molecularweight, relatively hydrophobic drugs to enhance solubility, reducetoxicity and alter biodistribution. Typically, PEGylated drugs areinjected as solutions.

A closely related application is synthesis of crosslinked degradable PEGnetworks or formulations for use in drug delivery since much of the samechemistry used in design of degradable, soluble drug carriers can alsobe used in design of degradable gels (Sawhney et al., Macromolecules 26:581-87, 1993). It also is known that intermacromolecular complexes canbe formed by mixing solutions of two complementary polymers. Suchcomplexes are generally stabilized by electrostatic interactions(polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) betweenthe polymers involved, and/or by hydrophobic interactions between thepolymers in an aqueous surrounding (Krupers et al., Eur. Polym J.32:785-790, 1996). For example, mixing solutions of polyacrylic acid(PAAc) and polyethylene oxide (PEO) under the proper conditions resultsin the formation of complexes based mostly on hydrogen bonding.Dissociation of these complexes at physiologic conditions has been usedfor delivery of free drugs (i.e., non-PEGylated). In addition, complexesof complementary polymers have been formed from both homopolymers andcopolymers.

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, reaction of the polypeptidewith N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG,mPEG₂-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate,mPEG succinimidyl propionate, mPEG succinimidyl butanoate, mPEGcarboxymethyl 3-hydroxybutanoic acid succinimidyl ester,homobifunctional PEG-succinimidyl propionate, homobifunctional PEGpropionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEGhydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate,propionaldehyde PEG, mPEG butryaldehyde, branched mPEG₂ butyraldehyde,mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless”maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester,mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfonePEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (seee.g., Monfardini et al., Bioconjugate Chem. 6:62-69, 1995; Veronese etal., J. Bioactive Compatible Polymers 12:197-207, 1997; U.S. Pat. Nos.5,672,662; 5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337;5,122,614; 5,324,844; U.S. 5,446,090; U.S. Pat. Nos. 5,612,460;5,643,575; 5,766,581; 5,795,569; 5,808,096; 5,900,461; 5,919,455;5,985,263; 5,990,237; 6,113,906; U.S. 6,214,966; U.S. Pat. Nos.6,258,351; 6,340,742; 6,413,507; 6,420,339; 6,437,025; 6,448,369;6,461,802; 6,828,401; 6,858,736; U.S. 2001/0021763; U.S. 2001/0044526;U.S. 2001/0046481; U.S. 2002/0052430; U.S. 2002/0072573; U.S.2002/0156047; U.S. 2003/0114647; U.S. 2003/0143596; U.S. 2003/0158333;U.S. 2003/0220447; U.S. 2004/0013637; US 2004/0235734; WO05000360; U.S.2005/0114037; U.S. 2005/0171328; U.S. 2005/0209416; EP 1064951; EP0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024; and WO0187925).

In one example, the polyethylene glycol has a molecular weight rangingfrom about 3 kD to about 50 kD, and typically from about 5 kD to about30 kD. Covalent attachment of the PEG to the drug (known as“PEGylation”) can be accomplished by known chemical synthesistechniques. For example, the PEGylation of protein can be accomplishedby reacting NHS-activated PEG with the protein under suitable reactionconditions.

While numerous reactions have been described for PEGylation, those thatare most generally applicable confer directionality, utilize mildreaction conditions, and do not necessitate extensive downstreamprocessing to remove toxic catalysts or bi-products. For instance,monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, and thusits use limits some of the heterogeneity of the resulting PEG-proteinproduct mixture. Activation of the hydroxyl group at the end of thepolymer opposite to the terminal methoxy group is generally necessary toaccomplish efficient protein PEGylation, with the aim being to make thederivatised PEG more susceptible to nucleophilic attack. The attackingnucleophile is usually the epsilon-amino group of a lysyl residue, butother amines also can react (e.g., the N-terminal alpha-amine or thering amines of histidine) if local conditions are favorable. A moredirected attachment is possible in proteins containing a single lysineor cysteine. The latter residue can be targeted by PEG-maleimide forthiol-specific modification. Alternatively, PEG hydrazide can be reactedwith a periodate oxidized hyaluronan-degrading enzyme and reduced in thepresence of NaCNBH₃. More specifically, PEGylated CMP sugars can bereacted with a hyaluronan-degrading enzyme in the presence ofappropriate glycosyl-transferases. One technique is the “PEGylation”technique where a number of polymeric molecules are coupled to thepolypeptide in question. When using this technique, the immune systemhas difficulties in recognizing the epitopes on the polypeptide'ssurface responsible for the formation of antibodies, thereby reducingthe immune response. For polypeptides introduced directly into thecirculatory system of the human body to give a particular physiologicaleffect (i.e., pharmaceuticals) the typical potential immune response isan IgG and/or IgM response, while polypeptides which are inhaled throughthe respiratory system (i.e., industrial polypeptide) potentially cancause an IgE response (i.e., allergic response). One of the theoriesexplaining the reduced immune response is that the polymeric molecule(s)shield(s) epitope(s) on the surface of the polypeptide responsible forthe immune response leading to antibody formation. Another theory or atleast a partial factor is that the heavier the conjugate is, the morereduced the resulting immune response is.

Typically, to make the PEGylated PH20 polypeptide provided herein, PEGmoieties are conjugated, via covalent attachment, to the polypeptides.Techniques for PEGylation include, but are not limited to, specializedlinkers and coupling chemistries (see e.g., Roberts, Adv. Drug Deliv.Rev. 54:459-476, 2002), attachment of multiple PEG moieties to a singleconjugation site (such as via use of branched PEGs; see e.g., Guiotto etal., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specificPEGylation and/or mono-PEGylation (see e.g., Chapman et al., NatureBiotech. 17:780-783, 1999), and site-directed enzymatic PEGylation (seee.g., Sato, Adv. Drug Deliv. Rev., 54:487-504, 2002). Methods andtechniques described in the art can produce proteins having 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to asingle protein molecule (see e.g., U.S. 2006/0104968).

As an exemplary illustrative method for making a PEGylated PH20polypeptide, PEG aldehydes, succinimides and carbonates have each beenapplied to conjugate PEG moieties, typically succinimidyl PEGs, torHuPH20. For example, rHuPH20 has been conjugated with exemplarysuccinimidyl monoPEG (mPEG) reagents including mPEG-SuccinimidylPropionates (mPEG-SPA), mPEG-Succinimidyl Butanoates (mPEG-SBA), and(for attaching “branched” PEGs) mPEG2-N-Hydroxylsuccinimide. ThesePEGylated succinimidyl esters contain different length carbon backbonesbetween the PEG group and the activated cross-linker, and either asingle or branched PEG group. These differences can be used, forexample, to provide for different reaction kinetics and to potentiallyrestrict sites available for PEG attachment to rHuPH20 during theconjugation process.

Succinimidyl PEGs (as above) containing either linear or branched PEGscan be conjugated to PH20. PEGs can used to generate PH20s reproduciblycontaining molecules having, on the average, between about three to sixor three to six PEG molecules per hyaluronidase. Such PEGylated rHuPH20compositions can be readily purified to yield compositions havingspecific activities of approximately 25,000 or 30,000 Unit/mg proteinhyaluronidase activity, and being substantially free of non-PEGylatedPH20 (less than 5% non-PEGylated).

Using various PEG reagents, exemplary versions of a PEGylated PH20polypeptide can be prepared, for example, using mPEG-SBA (30 kD),mPEG-SMB (30 kD), and branched versions based on mPEG2-NHS (40 kD) andmPEG2-NHS (60 kD). PEGylated versions of PH20 can be generated using NHSchemistries, as well as carbonates, and aldehydes, using each of thefollowing reagents: mPEG2-NHS-40K branched, mPEG-NHS-10K branched,mPEG-NHS-20K branched, mPEG2-NHS-60K branched; mPEG-SBA-5K,mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB-20K, mPEG-SMB-30K;mPEG-butyrldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin.PEGylated PH20 also can be prepared using PEG reagents available fromDowpharma, a division of Dow Chemical Corporation; including PH20polypeptides PEGylated with Dowpharma's p-nitrophenyl-carbonate PEG (30kDa) and with propionaldehyde PEG (30 kDa).

In one example, the PEGylation includes conjugation of mPEG-SBA, forexample, mPEG-SBA-30K (having a molecular weight of about 30 kDa) oranother succinimidyl ester of a PEG butanoic acid derivative, to a PH20polypeptide. Succinimidyl esters of PEG butanoic acid derivatives, suchas mPEG-SBA-30K readily couple to amino groups of proteins. For example,covalent conjugation of m-PEG-SBA-30K and rHuPH20 (which isapproximately 60 KDa in size) provides stable amide bonds betweenrHuPH20 and mPEG, as shown in Scheme 1, below.

Typically, the mPEG-SBA-30K or other PEG is added to the PH20polypeptide at a PEG:polypeptide molar ratio of 10:1 in a suitablebuffer, e.g., 130 mM NaCl/10 mM HEPES at pH 6.8 or 70 mM phosphatebuffer, pH 7, followed by sterilization, e.g., sterile filtration, andcontinued conjugation, for example, with stirring, overnight at 4° C. ina cold room. In one example, the conjugated PEG-PH20 is concentrated andbuffer-exchanged.

Other methods of coupling succinimidyl esters of PEG butanoic acidderivatives, such as mPEG-SBA-30K are known in the art (see e.g., U.S.Pat. Nos. 5,672,662; 6,737,505; and U.S. 2004/0235734). For example, apolypeptide, such as a PH20 polypeptide, can be coupled to an NHSactivated PEG derivative by reaction in a borate buffer (0.1 M, pH 8.0)for one hour at 4° C. The resulting PEGylated protein can be purified byultrafiltration. Another method reacts polypeptide with mPEG-SBA indeionized water to which triethylamine is added to raise the pH to7.2-9. The resulting mixture is stirred at room temperature for severalhours to complete the PEGylation.

Methods for PEGylation of PH20 polypeptides, including, for example,animal-derived hyaluronidases and bacterial hyaluronan-degradingenzymes, are known to one of skill in the art. See, for example,European Patent No. EP 0400472, which describes the PEGylation of bovinetestes hyaluorindase and chondroitin ABC lyase. Also, U.S. PublicationNo. 2006014968 describes PEGylation of a human hyaluronidase derivedfrom human PH20. For example, the PEGylated hyaluronan-degrading enzymegenerally contains at least 3 PEG moieties per molecule. In someexamples, the PH20 polypeptide contains three to six PEG molecules. Inother examples, the enzyme can have a PEG to protein molar ratio between5:1 and 9:1, for example, 7:1.

F. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS, DOSAGES ANDADMINISTRATION

Pharmaceutical compositions of any of the modified PH20 polypeptides areprovided herein for administration. Pharmaceutically acceptablecompositions are prepared in view of approvals for a regulatory agencyor other agency prepared in accordance with generally recognizedpharmacopeia for use in animals and in humans. Typically, the compoundsare formulated into pharmaceutical compositions using techniques andprocedures well known in the art (see e.g., Ansel Introduction toPharmaceutical Dosage Forms, Fourth Edition, 1985, 126).

In particular, provided herein are pharmaceutical compositions that arestable as a liquid formulation for prolonged periods of time for atleast 1 month at temperatures from or from about 2° C. to 8° C.,inclusive or for at least 3 days at a temperature from or from about 30°C. to 42° C., inclusive. Pharmaceutical compositions, in particularliquid formulations, can be limited by the stability of the activeagent, which can be susceptible to effects of storage conditions (timeor length of storage, temperature and/or agitation) and/or formulationcomponents contained in the composition. Hence, the stablepharmaceutical compositions generally contain a modified PH20polypeptide as described in Section C.1.b that exhibits increasedstability manifested as an increased resistance to one or more proteindenaturation conditions. Such protein denaturation conditions caninclude, but are not limited to, elevated temperature greater than orequal to or about 30° C., agitation, low or no salt, and presence ofexcipients. The increased stability is characterized by improved storagetime, decreased fragmentation, and/or decreased aggregate formation,while still retaining the activity of the active agent(s), e.g., thePH20 hyaluronidase. Such formulations can be provided as “ready-to use”liquid formulations without further reconstitution and/or without anyrequirement for further dilution. In some examples, the formulationsalso can be prepared in a lyophilized or concentrated form.

Pharmaceutical compositions containing a modified PH20 polypeptide canbe co-administered with another therapeutic agent. In such examples, themodified PH20 polypeptides can be formulated separately as apharmaceutical composition and administered prior to, simultaneouslywith, intermittently with, or subsequent to a second compositioncontaining an active therapeutic agent. In other examples, modified PH20polypeptides can be co-formulated with pharmaceutical formulations ofother therapeutic agents.

In particular, provided herein are co-formulations containing a modifiedPH20 polypeptide as described herein and a therapeutic agent that is achemotherapeutic agent, an analgesic agent, an anti-inflammatory agent,an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent,an anti-Parkinson agent, an anti-malarial agent, an anticonvulsantagent, an anti-depressant agent, and antiarthritics agent, ananti-fungal agent, an antihypertensive agent, an antipyretic agent, ananti-parasite agent, an antihistamine agent, an alpha-adrenergic agonistagent, an alpha blocker agent, an anesthetic agent, a bronchial dilatoragent, a biocide agent, a bactericide agent, a bacteriostat agent, abeta adrenergic blocker agent, a calcium channel blocker agent, acardiovascular drug agent, a contraceptive agent, a decongestant agent,a diuretic agent, a depressant agent, a diagnostic agent, a electrolyteagent, a hypnotic agent, a hormone agent, a hyperglycemic agent, amuscle relaxant agent, a muscle contractant agent, an ophthalmic agent,a parasympathomimetic agent, a psychic energizer agent, a sedativeagent, a sympathomimetic agent, a tranquilizer agent, an urinary agent,a vaginal agent, a viricide agent, a vitamin agent, a non-steroidalanti-inflammatory agent, an angiotensin converting enzyme inhibitoragent, a polypeptide, a protein, a nucleic acid, a drug, an organicmolecule or a sleep inducer. For example, modified PH20 polypeptidesprovided herein can be co-formulated with an antibody such as amonoclonal antibody, an Immune Globulin, an antibiotic, abisphosphonate, a cytokine, a chemotherapeutic agent, a coagulationfactor or an insulin. Exemplary therapeutic agents that can beco-formulated with a modified PH20 polypeptide are described indescribed in Section H. In particular, provided herein areco-formulations containing a modified PH20 polypeptide and an insulin,such as a fast-acting insulin, for example, a regular insulin or afast-acting (rapid-acting) insulin analog. The co-formulations providedherein include stable co-formulations, whereby the active agents, i.e.,the modified PH20 polypeptide and the therapeutic agent, exhibitincreased stability and retain activity for prolonged periods asdescribed herein.

Formulations containing PH20 provided herein, including separateformulations thereof and co-formulations, are stable for prolongedperiods of time, including at varied temperatures and under variedstorage or use conditions such as agitation. For example, theformulations provided herein are stable and retain activity of activeagent(s) (e.g., PH20 hyaluronidase) at “refrigerator” conditions, forexample, at 2° C. to 8° C., such as at or about 4° C., for at least atleast 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, atleast 8 months, at least 9 months, at least 10 months, at least 11months, at least 12 months, 13 months, 14 months, 15 months, 16 months,17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 monthsor 30 months or more. In another example, the formulations providedherein are stable and retain activity of active agent(s) (e.g., PH20hyaluronidase) at room temperature for example at 18° C. to 32° C.,generally 20° C. to 32° C., such as 28° C. to 32° C., for at least 2weeks to 1 year, for example, at least 3 weeks, 4 weeks, 2 months, 3months, 4 months, 5 months, 6 months, at least 7 months, at least 8months, at least 9 months, or at least 1 year or more. In a furtherexample, the formulations provided herein are stable and retain activityof active agent(s) (e.g., PH20 hyaluronidase) at elevated temperaturesof about or greater than 30° C., generally from or from about 30° C. to42° C., such as 32° C. to 37° C. or 35° C. to 37° C. or about or 37° C.for at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35days, 40 days, 45 days, 50 days, 60 days or more.

Compositions can take the form of solutions, suspensions, emulsions,tablets, pills, capsules, powders, and sustained release formulations. Acomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides. Oral formulation can includestandard carriers such as pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, and other such agents. Topical formulations also arecontemplated. The formulation should suit the mode of administration.

1. Formulations—Liquids, Injectables and Emulsions

The formulation generally is made to suit the route of administration.Parenteral administration, generally characterized by injection orinfusion, either subcutaneously, intramuscularly, intravenously orintradermally is contemplated herein. Preparations for parenteraladministration include sterile solutions ready for injection, steriledry soluble products, such as lyophilized powders, ready to be combinedwith a solvent just prior to use, including hypodermic tablets, sterilesuspensions ready for injection, sterile dry insoluble products ready tobe combined with a vehicle just prior to use and sterile emulsions.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. For example,the compositions containing a modified PH20 polypeptide, formulatedseparately or co-formulated with another therapeutic agent, can beprovided as a pharmaceutical preparation in liquid form as a solution,syrup or suspension. In liquid form, the pharmaceutical preparations canbe provided as a concentrated preparation to be diluted to atherapeutically effective concentration before use. Generally, thepreparations are provided in a dosage form that does not requiredilution for use. In another example, pharmaceutical preparations can bepresented in lyophilized form for reconstitution with water or othersuitable vehicle before use.

Injectables are designed for local and systemic administration. Forpurposes herein, local administration is desired for directadministration to the affected interstitium. The solutions can be eitheraqueous or nonaqueous. If administered intravenously, suitable carriersinclude physiological saline or phosphate buffered saline (PBS), andsolutions containing thickening and solubilizing agents, such asglucose, polyethylene glycol, and polypropylene glycol and mixturesthereof.

The concentration of the pharmaceutically active compound is adjusted sothat an injection or infusion provides an effective amount to producethe desired pharmacological effect. The exact dose depends on the age,weight and condition of the patient or animal as is known in the art.The unit-dose parenteral preparations can be packaged in, for example,an ampoule, a cartridge, a vial or a syringe with a needle. The volumeof liquid solution or reconstituted powder preparation, containing thepharmaceutically active compound, is a function of the disease to betreated and the particular article of manufacture chosen for package.All preparations for parenteral administration must be sterile, as isknown and practiced in the art. The percentage of active compoundcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the compound and theneeds of the subject.

Pharmaceutical compositions can include carriers or other excipients.For example, pharmaceutical compositions provided herein can contain anyone or more of a diluents(s), adjuvant(s), antiadherent(s), binder(s),coating(s), filler(s), flavor(s), color(s), lubricant(s), glidant(s),preservative(s), detergent(s), sorbent(s) or sweetener(s) and acombination thereof or vehicle with which a modified PH20 polypeptide isadministered. For example, pharmaceutically acceptable carriers orexcipients used in parenteral preparations include aqueous vehicles,nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers,antioxidants, local anesthetics, suspending and dispersing agents,emulsifying agents, sequestering or chelating agents and otherpharmaceutically acceptable substances. Formulations, including liquidpreparations, can be prepared by conventional means withpharmaceutically acceptable additives or excipients.

Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the compound,generally in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Suchpharmaceutical carriers can be sterile liquids, such as water or oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is atypical carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Examples of aqueous vehicles include SodiumChloride Injection, Ringers Injection, Isotonic Dextrose Injection,Sterile Water Injection, Dextrose and Lactated Ringers Injection.Nonaqueous parenteral vehicles include fixed oils of vegetable origin,cottonseed oil, corn oil, sesame oil and peanut oil. Suspending anddispersing agents include, but are not limited to, sorbitol syrup,cellulose derivatives or hydrogenated edible fats, sodiumcarboxymethylcellulose, hydroxypropyl methylcellulose andpolyvinylpyrrolidone. Emulsifying agents include, but are not limitedto, lecithin or acacia. Detergents include, but are not limited to,Polysorbate 80 (TWEEN 80). Non-aqueous vehicles include, but are notlimited to, almond oil, oily esters, or fractionated vegetable oils.Anti-microbial agents or preservatives include, but are not limited to,methyl or propyl-p-hydroxybenzoates or sorbic acid, m-cresol, phenol. Adiluent includes, but is not limited to, lactose, sucrose, dicalciumphosphate, or carboxymethylcellulose. A lubricant includes, but is notlimited to, magnesium stearate, calcium stearate or talc. A binderincludes, but is not limited to, starch, natural gums, such as gumacacia, gelatin, glucose, molasses, polyvinylpyrrolidine, celluloses andderivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Isotonic agents include, but are notlimited to, sodium chloride and dextrose. Buffers include, but are notlimited to, phosphate and citrate. Antioxidants include sodiumbisulfate. Local anesthetics include procaine hydrochloride. Asequestering or chelating agent of metal ions includes EDTA. Othersuitable pharmaceutical excipients include, but are not limited to,starch, glucose, lactose, dextrose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, saline, water,and ethanol. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles andsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pHadjustment. A composition, if desired, also can contain minor amounts ofnon-toxic auxiliary substances such as wetting or emulsifying agents, orpH buffering agents, for example, acetate, sodium citrate, cyclodextrinderivatives, sorbitan monolaurate, triethanolamine sodium acetate,triethanolamine oleate, stabilizers, solubility enhancers, and othersuch agents such as for example, sodium acetate, sodium phosphate,sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

In particular, antimicrobial agents (e.g., preservatives) inbacteriostatic or fungistatic concentrations (e.g., an anti-microbialeffective amount) can be added to parenteral preparations packaged inmultiple-dose containers, which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.

The volume of the formulations, including the separately formulated orco-formulated PH20-containing formulations provided herein, can be anyvolume suitable for the container in which it is provided. In someexamples, the formulations are provided in a vial, syringe, pen,reservoir for a pump or a closed loop system, or any other suitablecontainer. For example, the formulations provided herein are between orabout between 0.1 mL to 500 mL, such as 0.1 mL to 100 mL, 1 mL to 100mL, 0.1 mL to 50 mL, such as at least or about at least or about or 0.1mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, 50mL or more.

a. Lyophilized Powders

Of interest herein are lyophilized powders, which can be reconstitutedfor administration as solutions, emulsions and other mixtures. They mayalso be reconstituted and formulated as solids or gels.

The sterile, lyophilized powder is prepared by dissolving a compound ofenzyme in a buffer solution. The buffer solution may contain anexcipient which improves the stability or other pharmacologicalcomponent of the powder or reconstituted solution, prepared from thepowder. Subsequent sterile filtration of the solution followed bylyophilization under standard conditions known to those of skill in theart provides the desired formulation. A liquid formulation as describedherein above can be prepared. The resulting mixture is sterile filteredor treated to remove particulates and to insure sterility, andapportioned into vials for lyophilization. For example, the lyophilizedpowder can be prepared by dissolving an excipient, such as dextrose,sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose orother suitable agent, in a suitable buffer, such as citrate, sodium orpotassium phosphate or other such buffer known to those of skill in theart. Then, a selected enzyme is added to the resulting mixture, andstirred until it dissolves.

Each vial is made to contain a single dosage or multiple dosages of thecompound. The lyophilized powder can be stored under appropriateconditions, such as at about 4° C. to room temperature. Reconstitutionof this lyophilized powder with an appropriate buffer solution providesa formulation for use in parenteral administration.

b. Exemplary Formulations

Single dose formulations of PH20 are known in the art. For example,Hylenex® recombinant hyaluronidase (hyaluronidase human injection)contains, per mL, 8.5 mg NaCl (145 mM), 1.4 mg dibasic sodium phosphate(9.9 mM), 1.0 mg human albumin, 0.9 mg edetate disodium (2.4 mM), 0.3 mgCaCl₂ (2.7 mM) and NaOH to adjust the pH to 7.4. Other formulations ofhuman soluble hyaluronidase, such as the rHuPH20 formulations describedin U.S. Pat. Pub. No. US2011/0053247, include 130 mM NaCl, 10 mM Hepes,pH 7.0; or 10 mM histidine, 130 mM NaCl, pH 6.0. Any of the modifiedPH20 polypeptides provided herein can be similarly formulated.

In addition to a therapeutically effective amount of a modified PH20polypeptide and/or other therapeutic agent, exemplary pharmaceuticalcompositions provided herein, including separately formulated- andco-formulated-PH20 containing formulations, can contain a concentrationof NaCl and are prepared at a requisite pH to maintain the stability ofthe active agent(s) (e.g., PH20 hyaluronidase and/or other co-formulatedtherapeutic agent). For multi-dose formulations and other formulationsstored for a prolonged time, the compositions generally also contain oneor more preservatives. Further stabilizing agents and other excipientsalso can be included. Exemplary components are described below.

i. Salt (e.g. NaCl)

In examples herein, the pharmaceutical compositions provided hereincontain a concentration of salt, such as sodium chloride (NaCl), tomaintain the stability of the active agent(s) (e.g., PH20hyaluronidase). Salt, such as NaCl, is generally required to retain PH20stability and activity. Low salt concentrations of generally less than120 mM can have deleterious effects on PH20 activity over time anddepending on temperature conditions. Hence, the absence of salt (e.g.NaCl) or a low concentration of salt (e.g. NaCl) can result ininstability of the protein. In some examples herein, however, modifiedPH20 polypeptides that exhibit increased stability in the absence of lowor no salt, such as low or no NaCl (see e.g., Section C.1.b.iii), arenot susceptible to denaturation. Also, the presence of salt (e.g. NaCl)can have differing effects on other therapeutic agents. For example, thesolubility of insulin and insulin analogs tends to increase with lowersalt concentration (e.g., <140 mM) and high salt concentrations canresult in crystallization/aggregation of insulin, especially at lowertemperatures (see e.g., U.S. Provisional Appl. No. 61/520,962; U.S.application Ser. Nos. 13/507,263 and 13/507,262; and International PCTApplication No. PCT/US2012/042816). Thus, pharmaceutical compositionsprovided herein are prepared in accordance with the requirements of theactive agent(s). It is within the level of one of skill in the art toassess the stability of the active agent(s) in the formulation and undervarious storage conditions (see e.g., Section G). In particular examplesherein, the pharmaceutical compositions, including the separatelyformulated or co-formulated PH20-containing formulations providedherein, contain NaCl at a concentration of between or about between 10mM to 200 mM, such as 10 mM to 50 mM, 50 mM to 200 mM, 50 mM to 120 mM,50 mM to 100 mM, 50 mM to 90 mM, 120 mM to 160 mM, 130 mM to 150 mM, 80mM to 140 mM, 80 mM to 120 mM, 80 mM to 100 mM, 80 mM to 160 mM, 100 mMto 140 mM, 120 mM to 120 mM or 140 mM to 180 mM.

ii. pH and Buffer

In examples herein, the pharmaceutical compositions provided herein areprepared at a pH to maintain the stability of the active agent(s) (e.g.,PH20 hyaluronidase). For example, the pharmaceutical compositionsprovided herein are prepared at a pH of between or about between 6.5 to7.8 such as between or about between 6.5 to 7.2, 7.0 to 7.8, 7.0 to 7.6or 7.2 to 7.4. Reference to pH herein is based on measurement of pH atroom temperature. It is understood that the pH can change during storageover time, but typically will remain between or between about pH 6.5 toor to about 7.8. For example, the pH can vary by ±0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5 or more. Exemplaryco-formulations provided herein have a pH of or of about 7.0±0.2,7.1±0.2, 7.2±0.2, 7.3±0.2, 7.4±0.2, 7.5±0.2 or 7.6±0.2 when prepared. Ifnecessary, pH can be adjusted using acidifying agents to lower the pH oralkalizing agents to increase the pH. Exemplary acidifying agentsinclude, but are not limited to, acetic acid, citric acid, sulfuricacid, hydrochloric acid, monobasic sodium phosphate solution, andphosphoric acid. Exemplary alkalizing agents include, but are notlimited to, dibasic sodium phosphate solution, sodium carbonate, orsodium hydroxide.

The compositions are generally prepared using a buffering agent thatmaintains the pH range. Any buffer can be used in formulations providedherein so long as it does not adversely affect the stability of theactive agent(s) (e.g., PH20 hyaluronidase), and supports the requisitepH range required. Examples of particularly suitable buffers includeTris, succinate, acetate, phosphate buffers, citrate, aconitate, malateand carbonate. Those of skill in the art, however, will recognize thatformulations provided herein are not limited to a particular buffer, solong as the buffer provides an acceptable degree of pH stability, or“buffer capacity” in the range indicated. Generally, a buffer has anadequate buffer capacity within about 1 pH unit of its pK (Lachman etal. In: The Theory and Practice of Industrial Pharmacy 3rd Edn.(Lachman, L., Lieberman, H A. and Kanig, J. L., Eds.), Lea and Febiger,Philadelphia, p. 458-460, 1986). Buffer suitability can be estimatedbased on published pK tabulations or can be determined empirically bymethods well known in the art. The pH of the solution can be adjusted tothe desired endpoint within the range as described above, for example,using any acceptable acid or base.

Buffers that can be included in the co-formulations provided hereininclude, but are not limited to, Tris (Tromethamine), histidine,phosphate buffers, such as dibasic sodium phosphate, and citratebuffers. Such buffering agents can be present in the co-formulations atconcentrations between or about between 1 mM to 100 mM, such as 10 mM to50 mM or 20 mM to 40 mM, such as at or about 30 mM. For example, suchbuffering agents can be present in the co-formulations in aconcentration of or about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM,19 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65mM, 70 mM, 75 mM, or more.

iii. Preservative(s)

In examples herein, multi-dose formulations or formulations stored forprolonged periods contain an anti-microbially effective amount ofpreservative or mixture of preservatives in an amount to have abacteriostatic or fungistatic effect. In particular examples, thepreservatives are present in a sufficient concentration to provide theanti-microbial requirements of, for example, the United StatesPharmacopoeia (USP) and the European Pharmacopoeia (EP), including theEP anti-microbial requirements (EPA) and the preferred EP anti-microbialrequirements (EPB) (see Table 4). Since the presence of preservatives,and in particular phenolic preservatives, can have deleterious effectson the stability of PH20, such formulations typically contain a modifiedPH20 polypeptide that exhibits increased stability in the presence ofpreservatives, such as any described in Section C.1.b.i herein.Generally, the amount maintains the stability of the active agent(s)(e.g., PH20 hyaluronidase).

An anti-microbial effective amount of preservative is an amount thatexhibits anti-microbial activity by killing or inhibiting thepropagation of microbial organisms in a sample of the composition asassessed in an antimicrobial preservative effectiveness test (APET). Oneof skill in the art is familiar with the antimicrobial preservativeeffectiveness test and standards to be meet under the USP and EPA or EPBin order to meet minimum requirements. In general, the antimicrobialpreservative effectiveness test involves challenging a composition withprescribed inoculums of suitable microorganisms, i.e., bacteria, yeastand fungi, storing the inoculated preparation at a prescribedtemperature, withdrawing samples at specified intervals of time andcounting the organisms in the sample (see, Sutton and Porter, (2002) PDAJournal of Pharmaceutical Science and Technology 56(4):300-311; TheUnited States Pharmacopeial Convention, Inc., (effective Jan. 1, 2002),The United States Pharmacopeia 25^(th) Revision, Rockville, Md., Chapter<51> Antimicrobial Effectiveness Testing; and European Pharmacopoeia,Chapter 5.1.3, Efficacy of Antimicrobial Preservation). Themicroorganisms used in the challenge generally include three strains ofbacteria, namely E. coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCCNo. 9027) and Staphylococcus aureus (ATCC No. 6538), yeast (Candidaalbicans ATCC No. 10231) and fungus (Aspergillus niger ATCC No. 16404),all of which are added such that the inoculated composition contains 10⁵or 10⁶ colony forming units (cfu) of microorganism per mL ofcomposition. The preservative properties of the composition are deemedadequate if, under the conditions of the test, there is a significantfall or no increase, as specified in Table 3 in the number ofmicroorganisms in the inoculated composition after the times and at thetemperatures prescribed. The criteria for evaluation are given in termsof the log reduction in the number of viable microorganism as comparedto the initial sample or the previous time point.

Non-limiting examples of preservatives that can be included in theco-formulations provided herein include, but are not limited to, phenol,meta-cresol (m-cresol), methylparaben, benzyl alcohol, thimerosal,benzalkonium chloride, 4-chloro-1-butanol, chlorhexidinedihydrochloride, chlorhexidine digluconate, L-phenylalanine, EDTA,bronopol (2-bromo-2-nitropropane-1,3-diol), phenylmercuric acetate,glycerol (glycerin), imidurea, chlorhexidine, sodium dehydroacetate,ortho-cresol (o-cresol), para-cresol (p-cresol), chlorocresol,cetrimide, benzethonium chloride, ethylparaben, propylparaben orbutylparaben and any combination thereof. For example, formulationsprovided herein can contain a single preservative. In other examples,the formulations contain at least two different preservatives or atleast three different preservatives. For example, formulations providedherein can contain two preservatives such as L-phenylalanine andm-cresol, L-phenylalanine and methylparaben, L-phenylalanine and phenol,m-cresol and methylparaben, phenol and methylparaben, m-cresol andphenol or other similar combinations. In one example, the preservativein the formulation contains at least one phenolic preservative. Forexample, the formulation contains phenol, m-cresol or phenol andm-cresol.

In the formulations provided herein, the total amount of the one or morepreservative agents as a percentage (%) of mass concentration (w/v) inthe formulation can be, for example, between from or between about from0.1% to 0.4%, such as 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%,0.1% to 0.2%, 0.2% to 0.3%, or 0.3% to 0.4%. Generally, the formulationscontain less than 0.4% (w/v) preservative. For example, theco-formulations provided herein contain at least or about at least 0.1%,0.12%, 0.125%, 0.13%, 0.14%, 0.15%, 0.16% 0.17%, 0.175%, 0.18%, 0.19%,0.2%, 0.25%, 0.3%, 0.325%, 0.35% but less than 0.4% total preservative.

In some examples, the formulations provided herein contain between orbetween about 0.1% to 0.25% phenol and between or about between 0.05% to0.2% m-cresol, such as between or about between 0.10% to 0.2% phenol andbetween or about between 0.06% to 0.18% m-cresol, or between or aboutbetween 0.1% to 0.15% phenol and between or about between 0.08% to 0.15%m-cresol. For example, formulations provided herein contain or containabout 0.1% phenol and 0.075% m-cresol; 0.1% phenol and 0.15% m-cresol;0.125% phenol and 0.075% m-cresol; 0.13% phenol and 0.075% m-cresol;0.13% phenol and 0.08% m-cresol; 0.15% phenol and 0.175% m-cresol; or0.17% phenol and 0.13% m-cresol.

iv. Stabilizers

In examples herein, the pharmaceutical compositions provided hereinoptionally can contain one or more other stabilizing agent to maintainthe stability of the active agent(s) (e.g., PH20 hyaluronidase).Included among the types of stabilizers that can be contained in theformulations provided herein are amino acids, amino acid derivatives,amines, sugars, polyols, salts and buffers, surfactants, and otheragents. The formulations provided herein contain at least onestabilizer. For example, the formulations provided herein contain atleast one, two, three, four, five, six or more stabilizers. Hence, anyone or more of an amino acids, amino acid derivatives, amines, sugars,polyols, salts and buffers, surfactants, and other agents can beincluded in the formulations herein. Generally, the formulations hereincontain at least contain a surfactant and an appropriate buffer.Optionally, the formulations provided herein can contain otheradditional stabilizers. Other components include, for example, one ormore tonicity modifiers, one or more anti-oxidation agents, or otherstabilizer.

Exemplary amino acid stabilizers, amino acid derivatives or aminesinclude, but are not limited to, L-Arginine, Glutamine, Glycine, Lysine,Methionine, Proline, Lys-Lys, Gly-Gly, Trimethylamine oxide (TMAO) orbetaine. Exemplary sugars and polyols include, but are not limited to,glycerol, sorbitol, mannitol, inositol, sucrose or trehalose. Exemplarysalts and buffers include, but are not limited to, magnesium chloride,sodium sulfate, Tris such as Tris (100 mM), or sodium Benzoate.Exemplary surfactants include, but are not limited to, poloxamer 188(e.g., Pluronic® F68), polysorbate 80 (PS80), polysorbate 20 (PS20).Other stabilizers include, but are not limited to, hyaluronic acid (HA),human serum albumin (HSA), phenyl butyric acid, taurocholic acid,polyvinylpyrolidone (PVP) or zinc.

In particular examples herein, the formulations contain one or moredetergents, such as surfactants, to maintain the stability of the activeagent(s) (e.g., PH20 hyaluronidase). For example, surfactants caninhibit aggregation of PH20 and minimize absorptive loss. Thesurfactants generally are non-ionic surfactants. Surfactants that can beincluded in the formulations herein include, but are not limited to,partial and fatty acid esters and ethers of polyhydric alcohols such asof glycerol, or sorbitol, poloxamers and polysorbates. For example,exemplary surfactants in the —formulations herein include any one ormore of poloxamer 188 (PLURONICS® poloxamer such as PLURONIC® F68poloxamer), TETRONICS® surfactant, polysorbate 20, polysorbate 80, PEG400, PEG 3000, Tween® surfactant (e.g., Tween® 20 surfactant or Tween®80 surfactant), Triton® X-100 surfactant, SPAN® surfactant, MYRJ®surfactant, BRIJ® surfactant, CREMOPHOR® surfactant, polypropyleneglycols or polyethylene glycols. In some examples, the formulationsherein contain poloxamer 188, polysorbate 20, polysorbate 80, generallypoloxamer 188 (pluronic F68). The formulations provided herein generallycontain at least one surfactant, such as 1, 2 or 3 surfactants.

In the formulations provided herein, the total amount of the one or moresurfactants as a percentage (%) of mass concentration (w/v) in theformulation can be, for example, between from or between about from0.005% to 1.0%, such as between from or between about from 0.01% to0.5%, such as 0.01% to 0.1% or 0.01% to 0.02%. Generally, theformulations contain at least 0.01% surfactant and contain less than1.0%, such as less than 0.5% or less than 0.1% surfactant. For example,the formulations provided herein can contain at or about 0.001%, 0.005%,0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%,0.055%, 0.06%, 0.065%, 0.07%, 0.08%, or 0.09% surfactant. In particularexamples, the formulations provided herein contain or contain about0.01% to or to about 0.05% surfactant.

Tonicity modifiers can be included in the formulation provided herein toproduce a solution with the desired osmolality. The formulationsprovided herein have an osmolality of between or about between 245mOsm/kg to 305 mOsm/kg. For example, the osmolality is or is about 245mOsm/kg, 250 mOsm/kg, 255 mOsm/kg, 260 mOsm/kg, 265 mOsm/kg, 270mOsm/kg, 275 mOsm/kg, 280 mOsm/kg, 285 mOsm/kg, 290 mOsm/kg, 295mOsm/kg, 300 mOsm/kg or 305 mOsm/kg. In some examples, the formulationshave an osmolality of or of about 275 mOsm/kg. Tonicity modifiersinclude, but are not limited to, glycerin, NaCl, amino acids,polyalcohols, trehalose, and other salts and/or sugars. The particularamount can be empirically determined in order to retain enzyme activity,and/or tonicity.

In other instances, glycerin (glycerol) is included in the formulations.For example, formulations provided herein typically contain less than 60mM glycerin, such as less than 55 mM, less than 50 mM, less than 45 mM,less than 40 mM, less than 35 mM, less than 30 mM, less than 25 mM, lessthan 20 mM, less than 15 mM, 10 mM or less. The amount of glycerintypically depends on the amount of NaCl present: the more NaCl presentin the formulation, the less glycerin is required to achieve the desiredosmolality or osmolarity. Thus, for example, in formulations containinghigher NaCl concentrations, little or no glycerin need be included inthe formulation. In contrast, in formulations containing slightly lowerNaCl concentrations, glycerin can be included. For example, formulationsprovided herein can contain glycerin at a concentration of 40 mM to 60mM, such as less than 50 mM, such as 20 mM to 50 mM, for example at orabout 50 mM.

The formulations provided herein also can contain antioxidants to reduceor prevent oxidation, in particular oxidation of the PH20 polypeptide.For example, oxidation can be effected by high concentrations ofsurfactant or hyaluronan oligomers. Exemplary antioxidants include, butare not limited to, cysteine, tryptophan and methionine. In particularexamples, the anti-oxidant is methionine. The formulations providedherein can include an antioxidant at a concentration from between orfrom about between 5 mM to or to about 50 mM, such as 5 mM to 40 mM, 5mM to 20 mM or 10 mM to 20 mM. For example, methionine can be providedin the formulations herein at a concentration from between or from aboutbetween 5 mM to or to about 50 mM, such as 5 mM to 40 mM, 5 mM to 20 mMor 10 mM to 20 mM. For example, an antioxidant, for example methionine,can be included at a concentration that is or is about 5 mM, 10 mM, 11mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 35mM, 40 mM, 45 mM or 50 mM. In some examples, the formulations contain 10mM to 20 mM methionine, such as or about 10 mM or 20 mM methionine.

The formulations provided herein also can contain an amino acidstabilizer, which contributes to the stability of the preparation. Thestabilizer can be a non-polar or basic amino acid. Exemplary non-polarand basic amino acids include, but are not limited to, alanine,histidine, arginine, lysine, ornithine, isoleucine, valine, methionine,glycine and proline. For example, the amino acid stabilizer is glycineor proline, typically glycine. The stabilizer can be a single amino acidor it can be a combination of 2 or more such amino acids. The amino acidstabilizers can be natural amino acids, amino acid analogues, modifiedamino acids or amino acid equivalents. Generally, the amino acid is anL-amino acid. For example, when proline is used as the stabilizer, it isgenerally L-proline. It is also possible to use amino acid equivalents,for example, proline analogues. The concentration of amino acidstabilizer, for example glycine, included in the formulation ranges from0.1 M to 1 M amino acid, typically 0.1 M to 0.75 M, generally 0.2 M to0.5 M, for example, at least at or about 0.1 M, 0.15 M, 0.2 M, 0.25 M,0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.6 M, 0.7 M, 0.75 M or more aminoacid. The amino acid, for example glycine, can be used in a form of apharmaceutically acceptable salt, such as hydrochloride, hydrobromide,sulfate, acetate, etc. The purity of the amino acid, for exampleglycine, should be at least 98%, at least 99%, or at least 99.5% ormore.

In examples herein, if necessary, hyaluronidase inhibitors are includedin a formulation to stabilize PH20, in particular to reduce the effectsof otherwise destabilizing agents and conditions, such as, for example,low salt, high pH, the presence of preservatives and elevatedtemperatures, present in the formulation. Such a component generally isnot required for pharmaceutical compositions containing a modified PH20polypeptide as provided herein that exhibits increased stability undersuch conditions. When provided, the hyaluronidase inhibitor is providedat least at its equilibrium concentration. One of skill in the art isfamiliar with various classes of hyaluronidase inhibitors (see e.g.,Girish et al. (2009) Current Medicinal Chemistry, 16:2261-2288, andreferences cited therein). One of skill in the art knows or candetermine by standard methods in the art the equilibrium concentrationof a hyaluronidase inhibitor in a reaction or stable composition herein.

An exemplary hyaluronidase inhibitor for use in the compositions hereinis hyaluronan (HA). Hyaluronic acid (HA, also known as hyaluronan andhyaluronate) is the natural substrate for PH20. HA is a non-sulfatedglycosaminoglycan that is widely distributed throughout connective,epithelial, and neural tissues. It is a polymer of up to 25,000disaccharide units, themselves composed of D-glucuronic acid andD-N-acetylglucosamine. The molecular weight of HA ranges from about 5kDa to 200,000 kDa. Any size HA can be used in the compositions as astabilizer. In some examples, the HA is a disaccharide, composed ofD-glucuronic acid and D-N-acetylglucosamine. In other examples, the HAis an oligosaccharide, such as a tetrasaccharide, containing 2 repeatingdisaccharide units, or alternatively, the HA used in the co-formulationsprovided herein can contain multiple repeating disaccharide units, suchas 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30 or more disaccharide units. In another example, the HA used in theformulations provided herein has a molecular weight that is from or fromabout 5 kDa to or to about 5,000 kDa; from or from about 5 kDa to or toabout 1,000 kDa; from or from about 5 kDa to or to about 500 kDa; orfrom or from about 5 kDa to or to about 200 kDa. Exemplary HAoligosaccharides for use in the formulations herein have a molecularweight of or of about 6.4 kDa, 74.0 kDa. or 234.4 kDa. The formulationscan contain 1 mg/mL to 20 mg/mL HA, 8 mg/mL to 12 mg/mL, such as atleast or about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL or 20 mg/mL ormore HA. In some examples, the molar ratio of HA to PH20 is or is about100,000:1, 95,000:1, 90,000:1, 85,000:1, 80,000:1, 75,000:1, 70,000:1,65,000:1, 60,000:1, 55,000:1, 50,000:1, 45,000:1, 40,000:1, 35,000:1,30,000:1, 25,000:1, 20,000:1, 15,000:1, 10,000:1, 5,000:1, 1,000:1,900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, or 100:1 orless.

In some examples, a nicotinic compound is used as a stabilizing agent.Nicotinic compounds include, but are not limited to, nicotinamide,nicotinic acid, niacin, niacinamide, vitamin B3 and/or salts thereofand/or any combination thereof. In particular applications, thestabilizing agent can include a nicotinic compound an amino acid oramino acids (see e.g., International Publication No. WO2010149772). Forexample, the amino acid can be arginine, glutamic acid and/or saltsthereof or combinations thereof.

2. Compositions for Other Routes of Administration

Depending upon the condition treated other routes of administration,such as topical application, transdermal patches, oral and rectaladministration are also contemplated herein.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories include solid bodies for insertion into the rectum whichmelt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The typical weight of a rectalsuppository is about 2 to 3 gm. Tablets and capsules for rectaladministration are manufactured using the same pharmaceuticallyacceptable substance and by the same methods as for formulations fororal administration. Formulations suitable for rectal administration canbe provided as unit dose suppositories. These can be prepared byadmixing the active compound with one or more conventional solidcarriers, for example, cocoa butter, and then shaping the resultingmixture.

For oral administration, pharmaceutical compositions can take the formof, for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well-known in the art.

Formulations suitable for buccal (sublingual) administration include,for example, lozenges containing the active compound in a flavored base,usually sucrose and acacia or tragacanth; and pastilles containing thecompound in an inert base such as gelatin and glycerin or sucrose andacacia.

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixtures can be solutions, suspensions,emulsion or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compounds or pharmaceutically acceptable derivatives thereof may beformulated as aerosols for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma). These formulations, foradministration to the respiratory tract, can be in the form of anaerosol or solution for a nebulizer, or as a microfine powder forinsufflation, alone or in combination with an inert carrier such aslactose. In such a case, the particles of the formulation will typicallyhave diameters of less than 50 microns, or less than 10 microns.

The compounds can be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients also can be administered.

Formulations suitable for transdermal administration are provided. Theycan be provided in any suitable format, such as discrete patches adaptedto remain in intimate contact with the epidermis of the recipient for aprolonged period of time. Such patches contain the active compound in anoptionally buffered aqueous solution of, for example, 0.1 to 0.2 Mconcentration with respect to the active compound. Formulations suitablefor transdermal administration also can be delivered by iontophoresis(see, e.g., Tyle, P, Pharmaceutical Research 3(6):318-326 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe active compound.

Pharmaceutical compositions also can be administered by controlledrelease formulations and/or delivery devices (see e.g., in U.S. Pat.Nos. 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,916,899; 4,008,719;4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,591,767; 5,639,476;5,674,533 and 5,733,566).

3. Dosages and Administration

The modified PH20 polypeptides provided herein can be formulated aspharmaceutical compositions for single dosage or multiple dosageadministration. The PH20 polypeptide is included in an amount sufficientto exert a therapeutically useful effect in the absence of undesirableside effects on the patient treated. The therapeutically effectiveconcentration can be determined empirically by testing the polypeptidesin known in vitro and in vivo systems such as by using the assaysprovided herein or known in the art (see e.g., Taliani et al., (1996)Anal. Biochem., 240: 60-67; Filocamo et al., (1997) J Virology, 71:1417-1427; Sudo, (1996) Antiviral Res. 32: 9-18; Bouffard et al., (1995)Virology, 209:52-59; Bianchi et al., (1996) Anal. Biochem., 237:239-244; Hamatake et al., (1996) Intervirology 39:249-258; Steinkuhleret al., (1998) Biochem., 37:8899-8905; D'Souza et al., (1995) J Gen.Virol., 76:1729-1736; Takeshita et al., (1997) Anal. Biochem.,247:242-246; see also e.g., Shimizu et al., (1994) J. Virol.68:8406-8408; Mizutani et al., (1996) J. Virol. 70:7219-7223; Mizutaniet al., (1996) Biochem. Biophys. Res. Commun., 227:822-826; Lu et al.(1996) Proc. Natl. Acad. Sci (USA), 93:1412-1417; Hahm et al., (1996)Virology, 226:318-326; Ito et al. (1996) J. Gen. Virol., 77:1043-1054;Mizutani et al. (1995) Biochem. Biophys. Res. Commun., 212:906-911; Choet al., (1997) J. Virol. Meth. 65:201-207) and then extrapolatedtherefrom for dosages for humans.

The amount of a modified PH20 to be administered for the treatment of adisease or condition can be determined by standard clinical techniques.In addition, in vitro assays and animal models can be employed to helpidentify optimal dosage ranges. The precise dosage, which can bedetermined empirically, can depend on the particular enzyme, the routeof administration, the type of disease to be treated and the seriousnessof the disease.

Hence, it is understood that the precise dosage and duration oftreatment is a function of the disease being treated and can bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values also can vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or use ofcompositions and combinations containing them. The compositions can beadministered hourly, daily, weekly, monthly, yearly or once. Generally,dosage regimens are chosen to limit toxicity. It should be noted thatthe attending physician would know how to and when to terminate,interrupt or adjust therapy to lower dosage due to toxicity, or bonemarrow, liver or kidney or other tissue dysfunctions. Conversely, theattending physician would also know how to and when to adjust treatmentto higher levels if the clinical response is not adequate (precludingtoxic side effects).

Typically, a therapeutically effective dose of a modified PH20 enzyme isat or about 10 Unit (U) to 500,000 Units, 100 Units to 100,000 Units,500 Units to 50,000 Units, 1000 Units to 10,000 Units, 5000 Units to7500 Units, 5000 Units to 50,000 Units, or 1,000 Units to 10,000 Units,generally 1,000 to 50,000 Units, in a stabilized solution or suspensionor a lyophilized form. For example, a PH20 polypeptide, can beadministered at a dose of at least or about at least or 10 U, 20 U, 30U, 40 U, 50 U, 100 U, 150 U, 200 U, 250 U, 300 U, 350 U, 400 U, 450 U,500 U, 600 U, 700 U, 800 U, 900 U, 1000 U, 2,000 U, 3,000 U, 4,000Units, 5,000 U or more. The formulations can be provided in unit-doseforms such as, but not limited to, ampoules, syringes and individuallypackaged tablets or capsules.

The PH20 enzyme can be administered alone, or with otherpharmacologically effective agent(s) or therapeutic agent(s), in a totalvolume of 0.1-100 mL, 1-50 mL, 10-50 mL, 10-30 mL, 1-20 mL, or 1-10 mL,typically 10-50 mL. Typically, volumes of injections or infusions of aPH20-containing composition are at least or at least about 0.01 mL, 0.05mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL or more.The formulations provided herein contain a modified PH20 polypeptide inan amount between or about between 30 Units/mL to 3000 U/mL, 300 U/mL to2000 U/mL or 600 U/mL to 2000 U/mL or 600 U/mL to 1000 U/mL, such as atleast or about at least 30 U/mL, 35 U/mL, 40 U/mL, 50 U/mL, 100 U/mL,200 U/mL, 300 U/mL, 400 U/mL, 500 U/mL, 600 U/mL, 700 U/mL, 800 U/mL,900 U/mL, 1000 U/mL, 2000 U/mL or 3000 U/mL. For example, theformulations provided herein contain a PH20 that is in an amount that isat least 100 U/mL to 1000 U/mL, for example at least or about at leastor about or 600 U/mL.

The PH20 polypeptide can be provided as a solution in an amount that isat least or about or is 100 U/mL, 150 U/mL, 200 U/mL, 300 U/mL, 400U/mL, 500 U/mL, 600 U/mL, 800 U/mL or 1000 U/mL, or can be provided in amore concentrated form, for example in an amount that is at least orabout or is 2000 U/mL, 3000 Units/mL, 4000 U/mL, 5000 U/mL, 8000 U/mL,10,000 U/mL or 20,000 U/mL for use directly or for dilution to theeffective concentration prior to use. The PH20 polypeptide compositionscan be provided as a liquid or lyophilized formulation.

When the PH20 is co-formulated with a therapeutic agent, dosages can beprovided as a ratio of the amount of a PH20 polypeptide to the amount oftherapeutic agent administered. For example, a PH20 polypeptide can beadministered at 1 hyaluronidase U/therapeutic agent U (1:1) to 50:1 ormore, for example, at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1,45:1, 50:1 or more.

The formulations provided herein, including co-formulations and/orstable formulations, can be prepared for single dose administration,multiple dose administration or continuous infusion administrations.Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see e.g., U.S. Pat. No.3,710,795), is also contemplated herein.

For example, formulations of pharmaceutically therapeutically activecompounds and derivatives thereof are provided for administration tohumans and animals in unit dosage forms or multiple dosage forms. Forexample, compounds can be formulated as tablets, capsules, pills,powders, granules, sterile parenteral solutions or suspensions, oralsolutions or suspensions, or oil-water emulsions containing suitablequantities of the compounds or pharmaceutically acceptable derivativesthereof. Each unit dose contains a predetermined quantity oftherapeutically active compound(s) sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit dose forms can be administered in fractions or multiples thereof. Amultiple dose form is a plurality of identical unit dosage formspackaged in a single container to be administered in segregated unitdose forms. Examples of multiple dose forms include vials, bottles oftablets or capsules or bottles of pints or gallons. Hence, multiple doseform is a multiple of unit doses that are not segregated in packaging.Generally, dosage forms or compositions containing active ingredient inthe range of 0.005% to 100% with the balance made up from non-toxiccarrier can be prepared.

Compositions provided herein typically are formulated for administrationby subcutaneous route, although other routes of administration arecontemplated, such as any route known to those of skill in the artincluding intramuscular, intraperitoneal, intravenous, intradermal,intralesional, intraperitoneal injection, epidural, vaginal, rectal,local, otic, transdermal administration or any route of administration.Formulations suited for such routes are known to one of skill in theart. Administration can be local, topical or systemic depending upon thelocus of treatment. Local administration to an area in need of treatmentcan be achieved by, for example, but not limited to, local infusionduring surgery, topical application, e.g., in conjunction with a wounddressing after surgery, by injection, by means of a catheter, by meansof a suppository, or by means of an implant. Compositions also can beadministered with other biologically active agents, either sequentially,intermittently or in the same composition.

The most suitable route in any given case depends on a variety offactors, such as the nature of the disease, the tolerance of the subjectto a particular administration route, the severity of the disease, andthe particular composition that is used. Typically, the compositionsprovided herein are administered parenterally. In some examples,modified PH20 polypeptide compositions are administered so that theyreach the interstitium of skin or tissues, thereby degrading theinterstitial space for subsequent delivery of a therapeutic agent. Thus,in some examples, direct administration under the skin, such as bysubcutaneous administration methods, is contemplated. Thus, in oneexample, local administration can be achieved by injection, such as froma syringe or other article of manufacture containing an injection devicesuch as a needle. In another example, local administration can beachieved by infusion, which can be facilitated by the use of a pump orother similar device. Other modes of administration also arecontemplated. For example, modified PH20 polypeptides, includedconjugated forms with increased half-life such as PEGylated formsthereof, can be administered intravenously. Pharmaceutical compositionscan be formulated in dosage forms appropriate for each route ofadministration.

Administration methods can be employed to decrease the exposure ofselected modified PH20 polypeptides to degradative processes, such asproteolytic degradation and immunological intervention via antigenic andimmunogenic responses. Examples of such methods include localadministration at the site of treatment. PEGylation of therapeuticsincreases resistance to proteolysis, increases plasma half-life, anddecreases antigenicity and immunogenicity. Examples of PEGylationmethodologies are known in the art (see for example, Lu and Felix, Int.J. Peptide Protein Res., 43: 127-138, 1994; Lu and Felix, Peptide Res.,6: 140-6, 1993; Felix et al., Int. J. Peptide Res., 46: 253-64, 1995;Benhar et al., J. Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., J.Immunol., 154: 3088-95, 1995; see also, Caliceti et al. (2003) Adv. DrugDeliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt2):3S-8S). PEGylation also can be used in the delivery of nucleic acidmolecules in vivo. For example, PEGylation of adenovirus can increasestability and gene transfer (see, e.g., Cheng e et al., (2003) Pharm.Res. 20(9): 1444-51).

Various other delivery systems are known and can be used to administerselected PH20 polypeptides, such as but not limited to, encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the compound, receptor mediated endocytosis, and delivery ofnucleic acid molecules encoding selected PH20 polypeptides such asretrovirus delivery systems.

Hence, in certain embodiments, liposomes and/or nanoparticles also canbe employed with administration of soluble PH20 polypeptides. Liposomesare formed from phospholipids that are dispersed in an aqueous mediumand spontaneously form multilamellar concentric bilayer vesicles (alsotermed multilamellar vesicles (MLVs)). MLVs generally have diameters offrom 25 nm to 4 μm. Sonication of MLVs results in the formation of smallunilamellar vesicles (SUVs) with diameters in the range of 200 to 500angstroms containing an aqueous solution in the core.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios of lipid to water, liposomes form. Physical characteristicsof liposomes depend on the pH, ionic strength and the presence ofdivalent cations. Liposomes can show low permeability to ionic and polarsubstances, but at elevated temperatures undergo a phase transitionwhich markedly alters their permeability. The phase transition involvesa change from a closely packed, ordered structure, known as the gelstate, to a loosely packed, less-ordered structure, known as the fluidstate. This occurs at a characteristic phase-transition temperature andresults in an increase in permeability to ions, sugars and drugs.

Liposomes interact with cells via different mechanisms: endocytosis byphagocytic cells of the reticuloendothelial system such as macrophagesand neutrophils; adsorption to the cell surface, either by nonspecificweak hydrophobic or electrostatic forces, or by specific interactionswith cell-surface components; fusion with the plasma cell membrane byinsertion of the lipid bilayer of the liposome into the plasma membrane,with simultaneous release of liposomal contents into the cytoplasm; andby transfer of liposomal lipids to cellular or subcellular membranes, orvice versa, without any association of the liposome contents. Varyingthe liposome formulation can alter which mechanism is operative,although more than one can operate at the same time. Nanocapsules cangenerally entrap compounds in a stable and reproducible way. To avoidside effects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) should be designed using polymers ableto be degraded in vivo. Biodegradable polyalkyl-cyanoacrylatenanoparticles that meet these requirements are contemplated for useherein, and such particles can be easily made.

4. Exemplary PH20-Insulin Co-Formulations Provided herein are stableco-formulations of a fast acting insulin, such as a rapid acting(fast-acting) insulin analog, and a modified PH20 polypeptide. Any ofthe modified PH20 polypeptides provided herein can be included in aco-formulation with insulin, such as any of the co-formulationsdescribed in U.S. application Ser. No. 13/507,263 or 13/507,262 or inInternational PCT Application Serial No. PCT/US2012/042816.

In particular, the modified PH20 polypeptide is a modified PH20polypeptide that exhibits increased stability under denaturationconditions, such as any set forth in Sections C.1.b. In particular, thePH20 polypeptide is a modified PH20 polypeptide that exhibits increasedstability to one or more phenolic preservatives, such as any set forthin Section C.1.b.i. For example, the PH20 polypeptide is a modified PH20polypeptide that contains an amino acid replacement with P at a positioncorresponding to position 204 with reference to amino acid positions setforth in SEQ ID NO:3, such as F204P with reference to any of SEQ ID NOs:3, 7 or 32-66. In other examples, the PH20 polypeptide is a modifiedPH20 polypeptide that contains an amino acid replacement with R at aposition corresponding to position 58 with reference to amino acidpositions set forth in SEQ ID NO:3, such as V58R with reference to anyof SEQ ID NOs: 3, 7 or 32-66.

The fast acting insulin can be a regular insulin or a rapid acting(fast-acting) insulin analog. Insulin is a polypeptide that whenprocessed is composed of 51 amino acids containing an A- and B-chain.Generally, insulin contains an A-chain of about 21 amino acids and aB-chain of about 30 amino acids. The A- and B-chains are linked bydisulfide bridges. Exemplary regular insulins include, for example, ahuman insulin (with an A chain having a sequence of amino acids setforth in SEQ ID NO:862 and a B chain having a sequence of amino acidsset forth in SEQ ID NO:863) or a porcine insulin (with an A chain havinga sequence of amino acids set forth as amino acid residue positions88-108 of SEQ ID NO:864 and a B chain having a sequence of amino acidsset forth as amino acid residue positions 25-54 of SEQ ID NO:864).Exemplary fast-acting insulin analogs are insulin variants that containone or more amino acid modifications compared to a human insulin setforth in SEQ ID NO: 862 and 863 (A and B chains). For example, exemplaryinsulin analogs are known to one of skill in the art, and include, butare not limited to, glulisine having an A-chain set forth in SEQ IDNO:862 and a B-chain that is a variant of SEQ ID NO:863 (B-chain; LysB3,GluB29), HMR-1 153 having an A-chain set forth in SEQ ID NO:862 and aB-chain that is a variant of SEQ ID NO:863 (B-chain; LysB3, IleB28),insulin aspart having an A-chain set forth in SEQ ID NO:862 and aB-chain that is a variant of SEQ ID NO:863 (B-chain; AspB28), andinsulin lispro having an A-chain set forth in SEQ ID NO:862 and aB-chain that is a variant of SEQ ID NO:863 (B-chain; LysB28, ProB29). Inevery instance above, the nomenclature of the analogs is based on adescription of the amino acid substitution at specific positions on theA or B chain of insulin, numbered from the N-terminus of the chain, inwhich the remainder of the sequence is that of natural human insulin.Exemplary of such analog forms, are set forth in SEQ ID NO:862 (A-chain)and having a B-chain set forth in any of SEQ ID NOs: 865-867.

The co-formulations are stable as a liquid formulation for prolongedperiods of time for at least 1 month at temperatures from or from about2° C. to 8° C., inclusive, or for at least 3 days at a temperature fromor from about 30° C. to 42° C., inclusive. For example, theco-formulations are stable and retain activity of the PH20 hyaluronidaseand insulin at “refrigerator” conditions, for example, at 2° C. to 8°C., such as at or about 4° C., for at least at least 2 months, 3 months,4 months, 5 months, 6 months, or 7 months, at least 8 months, at least 9months, at least 10 months, at least 11 months, at least 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, 24 months, 25months, 26 months, 27 months, 28 months, 29 months or 30 months or more.In another example, the formulations provided herein are stable andretain activity of the PH20 hyaluronidase and insulin at roomtemperature for example at 18° C. to 32° C., generally 20° C. to 32° C.,such as 28° C. to 32° C., for at least 2 weeks to 1 year, for example,at least 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6months, at least 7 months, at least 8 months, at least 9 months, or atleast 1 year or more. In a further example, the formulations providedherein are stable and retain activity of the PH20 hyaluronidase andinsulin at elevated temperatures of about or greater than 30° C.,generally from or from about 30° C. to 42° C., such as 32° C. to 37° C.or 35° C. to 37° C. or about or 37° C. for at least 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50days, 60 days or more.

Assays to assess stability of active agents are well-known to one ofskill in the art. Section G provides exemplary assays to assessstability of PH20 hyaluronidase. The stability of insulin can beassessed using similar methods well-known to one of skill in the art.For example, insulin stability and solubility can be assessed by visualassessment (e.g., including changes in color, clarity, presence ofaggregates or clumping and material adhesion, or frosting), acidclarification, optical microscopy, reversed phase high performanceliquid chromatography (RP-HPLC), in vitro or in vivo bioassays anddenaturing and non-denaturing size exclusion chromatography (SEC). Invitro or in vivo bioassays for insulin activity include, but are notlimited to, a competitive binding assay using cells expressing insulinreceptors (e.g., human placental cell membranes) and a radiolabeledinsulin (see e.g., Weiss et al., (2001) J. Biol. Chem. 276:40018-40024;Duttaroy et al., (2005) Diabetes 54:251-258); insulin-stimulated glucoseuptake (Louveau et al., (2004) J. Endocrin. 181:271-280, Duttaroy etal., (2005) Diabetes 54:251-258); assays to assess glucose production inthe presence of insulin (Wang et al., (2000) J. Biochem.,275:14717-14721, Duttaroy et al., (2005) Diabetes 54:251-258); andstudies using diabetic and/or healthy animal models (Atkinson et al.,(1999) Nature Med. 5:601-604; Nagoya-Shibata-Yasuda (NSY) mice, Zuckerdiabetic fatty (ZDF) rats and Gato-Katazaki (GK) rats (Cefalu (2006)ILAR Journal 47:186-198).

Examples of such formulations contain 100 U/mL to 1000 U/mL of amodified PH20 polypeptide, and in particular at or about or at least 600U/mL; 10 U/mL to 1000 U/mL of a fast-acting insulin, and in particularat or at least or about 100 U/mL; NaCl at a concentration of between orabout between 80-140 mM; a pH of between or about between 7.0 to 7.8; abuffering agent that maintains the pH range of between or about between7.0 to 7.8; 0.1% to 0.4% preservative as a mass concentration (w/v).Optionally, a further stabilizing agent can be included. For example,the co-formulations provided herein contain 1 mM to 100 mM of abuffering agent. For example, the co-formulations provided hereincontain 0.005% to 0.5% surfactant. Exemplary co-formulations providedherein also can contain less than 60 mM glycerin (glycerol) and 2 mM toor to about 50 mM of an antioxidant.

The following stable formulations are exemplary only and provide aplatform from which minor adjustments can be made. It is understood thatvery small changes in the concentrations of the various excipients andother components (e.g., ±15% of the stated concentrations), or smallchanges in pH, can be made while retaining some if not all of theinsulin solubility and stability and PH20 stability. Further changesalso can be made by adding or removing excipients. For example, the typeof stabilizing surfactant can be changed.

For example, the exemplary co-formulations herein contain 100 U/mL to1000 U/mL of a modified PH20 polypeptide, and in particular at least orabout at least or about 600 U/mL of a modified PH20 polypeptide; 10 U/mLto 1000 U/mL of a fast-acting insulin, and in particular at least orabout at least or about 100 U/mL of a fast-acting insulin; from or fromabout 10 mM to or to about 50 mM Tris (e.g., from or from about 20 mM to40 mM Tris, such as or as about 20 mM, 25 mM, 30 mM, 35 mM or 40 mMTris); from or from about 80 mM to or to about 160 mM NaCl (e.g., at orabout 80 mM, 90 mM, 100 mM, 110 mM 120 mM, 130 mM, 140 mM, 150 mM or 160mM NaCl); from or from about 2 mM to or to about 50 mM methionine (e.g.,at or about 5 mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM methionine); fromor from about 0 mM to or to about 50 mM glycerin (e.g., at or about 5mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM glycerin); from or from about0.005% to or to about 0.5% poloxamer 188, such as 0.01% to 0.05% (e.g.,at or about 0.01%, 0.02%, 0.03%, 0.04% or 0.05% poloxamer 188); from orfrom about 0.05% to or to about 0.25% phenol (e.g., at or about 0.1%,0.12%, 0.125%, 0.13%, 0.14%, 0.15%, 0.16% or 0.17% phenol); and from orfrom about 0.05% to or to about 0.4% m-cresol (e.g., at or about 0.075%,0.08%, 0.09%, 0.1%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16% or 0.17%m-cresol). The formulations are prepared with a pH from or from about7.0 to or to about 7.6 (e.g., at or about pH 7.0, 7.1, 7.2, 7.3, 7.4,7.5 or 7.6). In further examples, zinc is included at a concentration ofor about 0.017 mg/100 U, 0.018 mg/100 U, 0.02 mg/100 U, 0.022 mg/100 Uor 0.024 mg/100 U insulin.

In particular examples, the fast acting insulin is insulin aspart,insulin lispro or insulin glulisine. Exemplary co-formulations providedherein that contain a modified PH20 polypeptide and insulin lispro arethose that contain from or about 25 mM to or to about 35 mM Tris (e.g.,at or about 30 mM Tris); from or from about 70 mM to or to about 100 mMNaCl (e.g., at or about 80 mM or 100 mM NaCl); from or from about 10 mMto or to about 30 mM methionine (e.g., at or about 10 mM or 20 mMmethionine); from or from about 40 mM to or to about 60 mM glycerin(e.g., at or about 50 mM glycerin); from or from about 0.005% to or toabout 0.05% poloxamer 188 (e.g., at or about 0.01% poloxamer 188); fromor from about 0.017 mg zinc/100 U insulin to or to about 0.024 mgzinc/100 U insulin (e.g., 0.017 mg zinc/100 U insulin, 0.018 mg/100 U,0.02 mg/100 U, 0.022 mg/100 U or 0.024 mg zinc/100 U insulin); from orfrom about 0.08% to or to about 0.17% phenol (e.g., 0.1%, 0.125% or0.13% phenol); and from or from about 0.07% to or to about 0.17%m-cresol (e.g., 0.075%, 0.08%, 0.13% or 0.15% m-cresol). For example,the co-formulations can contain at or about 0.1% phenol and 0.015%m-cresol; at or about 0.125% phenol and 0.075% m-cresol; at or about0.13% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.08%m-cresol; or at or about 0.17% phenol and 0.13% m-cresol. Suchformulations of insulin lispro and a modified PH20 polypeptide areprepared with a pH of or about 7.0 to or to about 7.5 (typically a pH ofor about pH 7.2).

Exemplary co-formulations provided herein that contain a modified PH20polypeptide and insulin aspart are those that contain from or from about25 mM to or to about 35 mM Tris (e.g., at or about 30 mM Tris); from orfrom about 70 mM to or to about 120 mM NaCl (e.g., at or about 80 mM or100 mM NaCl); from or from about 2 mM to or to about 30 mM methionine,such as 2 mM to 10 mM or 5 mM to 30 mM methionine (e.g., at or about 5mM, 10 mM or 20 mM methionine); from or from about 0.005% to or to about0.05% poloxamer 188 (e.g., at or about 0.01% poloxamer 188); from orfrom about 0.08% to or to about 0.17% phenol (e.g., 0.1%, 0.125% or0.13% phenol); and from or from about 0.07% to or to about 0.17%m-cresol (e.g., 0.075%, 0.08%, 0.13% or 0.15% m-cresol). For example,the co-formulations can contain at or about 0.1% phenol and 0.015%m-cresol; at or about 0.125% phenol and 0.075% m-cresol; at or about0.13% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.08%m-cresol; or at or about 0.17% phenol and 0.13% m-cresol. Suchformulations of insulin aspart and a modified PH20 polypeptide areprepared with a pH of or about 7.0 to or to about 7.6 (typically a pH ofor about pH 7.4 or 7.3).

Further exemplary formulations provided herein that contain a modifiedPH20 polypeptide and insulin aspart are those that do not containphenol. Such exemplary formulations contain from or from about 25 mM toor to about 35 mM Tris (e.g., at or about 30 mM Tris); from or fromabout 70 mM to or to about 120 mM NaCl (e.g., at or about 80 mM or 100mM NaCl); from or from about 2 mM to or to about 30 mM methionine, suchas 2 mM to 10 mM or 5 mM to 30 mM methionine (e.g., at or about 5 mM, 10mM or 20 mM methionine); from or from about 0.005% to or to about 0.05%poloxamer 188 (e.g., at or about 0.01% poloxamer 188); and from or fromabout 0.07% to or to about 0.4% m-cresol, such as from or from about0.2% to 0.4% m-cresol (e.g., 0.3%, 0.315%, 0.35%, 0.4% m-cresol). Suchformulations of insulin aspart and a modified PH20 polypeptide areprepared with a pH of or about 7.0 to or to about 7.6 (typically a pH ofor about pH 7.4 or 7.3).

Exemplary co-formulations provided herein that contain a modified PH20polypeptide and insulin glulisine are those that contain from or fromabout 25 mM to or to about 35 mM Tris (e.g., at or about 30 mM Tris);from or from about 100 mM to or to about 150 mM NaCl (e.g., at or about100 mM or 140 mM NaCl); from or from about 10 mM to or to about 30 mMmethionine (e.g., at or about 10 mM or 20 mM methionine); from or fromabout 40 mM to or to about 60 mM glycerin (e.g., at or about 50 mMglycerin); from or from about 0.005% to or to about 0.05% poloxamer 188(e.g., at or about 0.01% poloxamer 188); from or from about 0.08% to orto about 0.17% phenol (e.g., 0.1%, 0.125% or 0.13% phenol); and from orfrom about 0.07% to or to about 0.17% m-cresol (e.g., 0.075%, 0.08%,0.13% or 0.15% m-cresol). For example, the co-formulations can containat or about 0.1% phenol and 0.015% m-cresol; at or about 0.125% phenoland 0.075% m-cresol; at or about 0.13% phenol and 0.075% m-cresol; at orabout 0.13% phenol and 0.08% m-cresol; or at or about 0.17% phenol and0.13% m-cresol. Such formulations of insulin glulisine and a modifiedPH20 polypeptide are prepared with a pH of or about 7.0 to or to about7.6 (typically a pH of or about pH 7.4).

5. Packaging, Articles of Manufacture and Kits

Pharmaceutical compounds of modified PH20 polypeptides, or nucleic acidsencoding such polypeptides, or derivatives or variants thereof can bepackaged as articles of manufacture containing packaging material, apharmaceutical composition which is effective for treating a disease ordisorder, and a label that indicates that the pharmaceutical compositionor therapeutic molecule is to be used for treating the disease ordisorder. Combinations of a selected modified PH20 polypeptide, or aderivative or variant thereof and an therapeutic agent also can bepackaged in an article of manufacture.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment. The articles of manufacture can includea needle or other injection device so as to facilitate administration(e.g., sub-epidermal administration) for local injection purposes. Awide array of formulations of the compounds and compositions providedherein are contemplated including a modified PH20 polypeptide and atherapeutic agent, such as a fast-acting insulin, known to treat aparticular disease or disorder. The choice of package depends on thePH20 and/or therapeutic agent, and whether such compositions will bepackaged together or separately. In one example, the PH20 can bepackaged as a mixture with the therapeutic agent. In another example,the components can be packaged as separate compositions

Modified PH20 polypeptides, therapeutic agents and/or articles ofmanufacture thereof also can be provided as kits. Kits can include apharmaceutical composition described herein and an item foradministration provided as an article of manufacture. For example a PH20polypeptide can be supplied with a device for administration, such as asyringe, an inhaler, a dosage cup, a dropper, or an applicator. Thecompositions can be contained in the item for administration or can beprovided separately to be added later. The kit can, optionally, includeinstructions for application including dosages, dosing regimens andinstructions for modes of administration. Kits also can include apharmaceutical composition described herein and an item for diagnosis.For example, such kits can include an item for measuring theconcentration, amount or activity of the selected protease in a subject.

G. METHODS OF ASSESSING PH20 ACTIVITY AND STABILITY

Assays can be used to assess the stability and activity of the PH20polypeptides provided herein. The assays can be used to assess thehyaluronidase activity of the PH20 polypeptide under particularconditions, temperature, and/or over time. Such assays can be used, forexample, to determine the stability of the PH20 polypeptide underspecific denaturation conditions, including, but not limited to,elevated temperatures greater than or about or 30° C. (e.g., 30° C. to42° C. such as or about 37° C.), agitation, presence of excipients(e.g., preservative), or low or no NaCl (salt). For example, stabilityunder specific conditions can be monitored by assessing activity,solubility, and stability (e.g., formation of aggregates, etc.) in theabsence of exposure to the denaturation condition and then at varioustime points thereafter in the presence of the condition. Hence,stability can be assessed over time. Stability also can be assessed bycomparing any one or more of activity, solubility or aggregation in thepresence of one or more denaturation conditions compared to a native,wildtype or reference PH20 polypeptide. The assays also can be used makeminor adjustments to the formulations provided herein while retainingthe stability of both active agents.

1. Hyaluronidase Activity

The activity of a modified PH20 polypeptide can be assessed usingmethods well known in the art. For example, the USP XXII assay forhyaluronidase determines activity indirectly by measuring the amount ofundegraded hyaluronic acid, or hyaluronan, (HA) substrate remainingafter the enzyme is allowed to react with the HA for 30 min at 37° C.(USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention,Inc, Rockville, Md.). A Hyaluronidase Reference Standard (USP) orNational Formulary (NF) Standard Hyaluronidase solution can be used inan assay to ascertain the activity, in units, of any hyaluronidase. Inone example, activity is measured using a microturbidity assay. This isbased on the formation of an insoluble precipitate when hyaluronic acidbinds with a reagent that precipitates it, such as acidified serum orcetylpyridinium chloride (CPC). The activity is measured by incubatinghyaluronidase with sodium hyaluronate (hyaluronic acid) for a set periodof time (e.g., 10 minutes) and then precipitating the undigested sodiumhyaluronate with the addition of acidified serum or CPC. The turbidityof the resulting sample is measured at 640 nm after an additionaldevelopment period. The decrease in turbidity resulting fromhyaluronidase activity on the sodium hyaluronate substrate is a measureof hyaluronidase enzymatic activity.

In another example, hyaluronidase activity is measured using amicrotiter assay in which residual biotinylated hyaluronic acid ismeasured following incubation with hyaluronidase (see e.g., Frost andStern (1997) Anal. Biochem. 251:263-269, U.S. Pat. Publication No.20050260186). The free carboxyl groups on the glucuronic acid residuesof hyaluronic acid are biotinylated, and the biotinylated hyaluronicacid substrate is covalently coupled to a microtiter plate. Followingincubation with hyaluronidase, the residual biotinylated hyaluronic acidsubstrate is detected using an avidin-peroxidase reaction, and comparedto that obtained following reaction with hyaluronidase standards ofknown activity.

Other assays to measure hyaluronidase activity also are known in the artand can be used in the methods herein (see e.g., Delpech et al., (1995)Anal. Biochem. 229:35-41; Takahashi et al., (2003) Anal. Biochem.322:257-263).

Many hyaluronidase assays have been based upon the measurement of thegeneration of new reducing N-acetylamino groups (Bonner and Cantey,Clin. Chim. Acta 13:746-752, 1966), or loss of viscosity (De Salegui etal., Arch. Biochem. Biophys. 121:548-554, 1967) or turbidity (Doifnianand Ott, J. Biol. Chem. 172:367, 1948). With purified substrates all ofthese methods suffice for determination of the presence or absence ofendoglycosidase activity.

Substantially purified glycosaminoglycan substrates can also be used ina Gel Shift Assay. Glycosaminoglycans are mixed with recombinant PH20,such as a soluble PH20, to test for endoglycosidase activity thatresults in a shift in substrate mobility within the gel. Examples ofsuch substrates include, but are not limited to, chondroitin-4 and 6sulfate, dermatan sulfate, heparan-sulfate, which can be obtained fromSigma Chemical. Human umbilical cord Hyaluronan can be obtained fromICN. For example, each test substrate can be diluted to at or about 0.1mg/mL in a buffer range from pH 3.5-7.5. In such an exemplary assay, ator about 10 μl samples of purified soluble PH20 or conditioned mediafrom PH20 expressing cells can be mixed with at or about 90 μl of testsubstrate in desired buffer and incubated for 3 hours at 37° C.Following incubation, samples are neutralized with sample buffer (TrisEDTA pH 8.0, Bromophenol Blue and glycerol) followed by electrophoresis.Glycosaminoglycans can be detected using any method known in the art,for example, glycosaminoglycans can be detected by staining the gelsusing 0.5% Alcian Blue in 3% Glacial Acetic Acid overnight followed bydestaining in 7% Glacial Acetic Acid. Degradation is determined bycomparison of substrate mobility in the presence and absence of enzyme.

Hyaluronidase activity can also be detected by substrate gel zymography(Guentenhoner et al., (1992) Matrix 12:388-396). In this assay, a sampleis applied to an SDS-PAGE gel containing hyaluronic acid and theproteins in the sample separated by electrophoresis. The gel is thenincubated in an enzyme assay buffer and subsequently stained to detectthe hyaluronic acid in the gel. Hyaluronidase activity is visualized asa cleared zone in the substrate gel.

The ability of a PH20 polypeptide, including a modified PH20 polypeptideprovided herein, to act as a spreading or diffusing agent also can beassessed. For example, trypan blue dye can be injected subcutaneouslywith or without a PH20 polypeptide into the lateral skin on each side ofnude mice. The dye area is then measured, such as with a microcaliper,to determine the ability of the PH20 polypeptide to act as a spreadingagent (U.S. Pat. Pub. No. 20060104968).

The functional activity of a PH20 polypeptide can be compared and/ornormalized to a reference standard using any of these assays. This canbe done to determine what a functionally equivalent amount of a PH20polypeptide is. For example, the ability of a PH20 polypeptide to act asa spreading or diffusing agent can be assessed by injecting it into thelateral skin of mice with trypan blue, and the amount required toachieve the same amount of diffusion as, for example, 100 units of aHyaluronidase Reference Standard, can be determined. The amount of PH20polypeptide required is, therefore, functionally equivalent to 100hyaluronidase units.

2. Solubility

The solubility of a PH20 polypeptide can be determined by any methodknown to one of the skill in the art. One method for determiningsolubility is detergent partitioning. For example, a soluble PH20polypeptide can be distinguished, for example, by its partitioning intothe aqueous phase of a Triton® X-114 detergent solution at 37° C.(Bordier et al., (1981) J. Biol. Chem., 256:1604-1607).Membrane-anchored polypeptides, such as lipid-anchored hyaluronidases,including GPI-anchored hyaluronidases, will partition into thedetergent-rich phase, but will partition into the detergent-poor oraqueous phase following treatment with Phospholipase C. Phospholipase Cis an enzyme that cleaves the phospho-glycerol bond found inGPI-anchored proteins. Treatment with PLC will cause release ofGPI-linked proteins from the outer cell membrane.

3. Purity, Crystallization or Aggregation

The stability of a PH20 polypeptide provided herein also can be assessedusing other methods and assays known in the art. In addition toassessing stability based on hyaluronidase activity, stability can beassessed by visual inspection, percent recovery, protein purity andapparent melting temperature.

For example, protein purity can be measured by reversed phase highperformance liquid chromatography (RP-HPLC). Protein purity, asdetermined by RP-HPLC, is the percent of the main PH20 protein peakpresent, as compared to all of the protein species present. Thus,RP-HPLC, and similar methods known to one of skill in the art, canassess degradation of the enzyme. Protein purity can be assessed overtime. Protein purity also can be assessed in the presence of one or moredenaturation conditions and in varying amounts thereof. Percent recoveryalso can be determined as the relative percentage of the polypeptideunder various conditions (denaturation conditions, time of storage, modeof storage such as vessel or container, or other similar parameters thatcan be altered) as compared to a reference sample. PH20 polypeptidestability also can be determined by measuring the oxidation of thehyaluronidase by RP-HPLC. Percent oxidation is a measure of sum of thepeak areas of the major (ox-1) and minor (ox-2) peaks.

In one example, the melting temperature of a PH20 polypeptide, such as amodified PH20 polypeptide, can be determined by measuring thehydrodynamic radius of particles by dynamic light scattering undervarious conditions (e.g., denaturation conditions or other storageconditions). An increase in particle size and a decrease in the meltingtemperature indicates denaturation and subsequent aggregation of thehyaluronidase.

Other methods known to one of skill in the art that can be used todetermine the stability of the hyaluronidase in the co-formulationsprovided herein, include polyacrylamide gel electrophoresis (PAGE),immunoblotting, nuclear magnetic resonance (NMR) spectroscopy, massspectrometry, circular dichroism (CD) and dye-based fluorescence assays.

4. Pharmacodynamics/Pharmacokinetics

The effect of administration of a PH20 polypeptide, such as a modifiedPH20 polypeptide, alone or in combination with another therapeuticagent, on the pharmacokinetic and pharmacodynamic properties of anyadministered agent also can be assessed in vivo using animal modelsand/or human subjects, such as in the setting of a clinical trial.Pharmacokinetic or pharmacodynamic studies can be performed using animalmodels or can be performed during studies with patients administeredwith a PH20 polypeptide or modified PH20 polypeptide.

Animal models include, but are not limited to, mice, rats, rabbits,dogs, guinea pigs and non-human primate models, such as cynomolgusmonkeys or rhesus macaques. In some instances, pharmacokinetic orpharmacodynamic studies are performed using healthy animals. In otherexamples, the studies are performed using animal models of a disease forwhich therapy with hyaluronan is considered, such as animal models ofany hyaluronan-associated disease or disorder, for example a tumormodel.

The pharmacokinetic properties of a PH20 polypeptide, such as a modifiedPH20 polypeptide, can be assessed by measuring such parameters as themaximum (peak) concentration (C_(max)), the peak time (i.e., whenmaximum concentration occurs; T_(max)), the minimum concentration (i.e.,the minimum concentration between doses; C_(max)), the eliminationhalf-life (T_(1/2)) and area under the curve (i.e., the area under thecurve generated by plotting time versus concentration; AUC), followingadministration. The absolute bioavailability of the hyaluronidase can bedetermined by comparing the area under the curve of hyaluronidasefollowing subcutaneous delivery (AUCsc) with the AUC of hyaluronidasefollowing intravenous delivery (AUC_(iv)). Absolute bioavailability (F),can be calculated using the formula:F=([AUC]_(sc)×dose_(sc))/([AUC]_(iv)×dose_(iv)). A range of doses anddifferent dosing frequency of dosing can be administered in thepharmacokinetic studies to assess the effect of increasing or decreasingconcentrations enzyme, such as modified PH20 polypeptide, in the dose.

H. METHODS OF TREATMENT AND COMBINATION THERAPY

Provided herein are methods and uses of any of the modified PH20polypeptides provided herein that exhibit hyaluronidase activity basedon its ability to degrade glycosaminoglycan(s) such as hyaluronan. Dueto such activity, the modified PH20 polypeptides can be used as aspreading factor to increase the delivery and/or bioavailability ofsubcutaneously administered therapeutic agents. Delivery of anytherapeutic agent, including but not limited to, peptides, proteins,small molecule drugs, nucleic acids, or viruses can be facilitated orenhanced by co-administration with a modified PH20 polypeptide providedherein. For example, modified PH20 polypeptides can be used to increasethe delivery of therapeutic agents such as antibodies (e.g., monoclonalantibodies), cytokines, Immune Globulin, an Insulin, or coagulationfactors, to a desired locus, such as by increasing penetration ofchemotherapeutic agents into solid tumors. The modified PH20polypeptides also can be used to treat a hyaluronan-disease or disorderthat is characterized by an excess or accumulation of hyaluronan. Forexample, modified PH20 polypeptides provided herein can be used to fortreating a tumor; for treating glycosaminoglycan accumulation in thebrain; for treating a cardiovascular disorder; for treating anophthalmic disorder; for treating pulmonary disease; for treatingcellulite; and/or for treating a proliferative disorder.

Other methods and uses of a modified PH20 polypeptide include any thatare known to one of skill in the art. For example, various forms of PH20hyaluronidases have been prepared and approved for therapeutic use inhumans. For example, animal-derived hyaluronidase preparations includeVitrase® hyaluronidase (ISTA Pharmaceuticals), a purified ovinetesticular hyaluronidase, and Amphadase® hyaluronidase (AmphastarPharmaceuticals), a bovine testicular hyaluronidase. Hylenex®hyaluronidase (Halozyme Therapeutics) is a human recombinanthyaluronidase produced by genetically engineered Chinese Hamster Ovary(CHO) cells containing nucleic acid encoding for soluble rHuPH20 (seee.g., U.S. Pat. No. 7,767,429). Approved therapeutic uses forhyaluronidases include use as an adjuvant to increase the absorption anddispersion of other therapeutic agents for hypodermoclysis (subcutaneousfluid administration), and as an adjunct in subcutaneous urography forimproving resorption of radiopaque agents. In addition to theseindications, hyaluronidases can be used as a therapeutic or cosmeticagent for the treatment of additional diseases and conditions. Forexample, hyaluronidase is commonly used, for example, for peribulbarblock in local anesthesia prior ophthalmic surgery. The presence of theenzyme prevents the need for additional blocks and reduces the time tothe onset of akinesia (loss of eye movement). Peribulbar and sub-Tenon'sblock are the most common applications of hyaluronidase for ophthalmicprocedures. Hyaluronidase also can promote akinesia in cosmetic surgery,such as blepharoplasties and face lifts. It is understood that solublePH20 hyaluronidases provided herein, including esPH20 hyaluronidases,can be used in any method of treatment or combination therapy for whicha PH20 hyaluronidase is used (see e.g., U.S. Publication Nos.US20040268425; US20050260186; US20060104968; and U.S. application Ser.No. 12/381,844, published as U.S. Publication No. US20100074885;12/386,249, published as U.S. Publication No. US20090311237; 12/387,225,published as U.S. Publication No. US20090304665; and Ser. No.12/386,222, published as U.S. Publication No. US2010003238, eachincorporated by reference in their entirety).

Exemplary, non-limiting, methods and uses are described in the followingsubsections.

1. Methods of Delivering Therapeutic Agents

As noted above, hyaluronidase is a spreading or diffusing substance thatmodifies the permeability of connective tissue through the hydrolysis ofhyaluronic acid, a polysaccharide found in the intercellular groundsubstance of connective tissue, and of certain specialized tissues, suchas the umbilical cord and vitreous humor. When no spreading factor ispresent, materials injected subcutaneously, such as drugs, proteins,peptides and nucleic acid, spread very slowly. Co-injection withhyaluronidase, however, can cause rapid spreading. The rate of diffusionis proportional to the amount of enzyme, and the extent of diffusion isproportional to the volume of solution.

Modified PH20 polypeptides provided herein can be used to promote orenhance the delivery agents and molecules to any of a variety ofmammalian tissues in vivo. It can be used to facilitate the diffusionand, therefore, promote the delivery, of small molecule pharmacologicagents as well as larger molecule pharmacologic agents, such asproteins, nucleic acids and ribonucleic acids, and macromolecularcompositions than can contain a combination of components including, butnot limited to, nucleic acids, proteins, carbohydrates, lipids,lipid-based molecules and drugs (see e.g., U.S. Publication Nos.US20040268425; US20050260186; and US20060104968). Modified PH20polypeptides can be co-administered and/or co-formulated with atherapeutic agent to improve the bioavailability as well aspharmacokinetic (PK) and/or pharmacodynamic (PD) characteristics ofco-formulated or co-administered agents. PK/PD parameters that can beimproved by using soluble PH20, such as esPH20, include such measures asC_(max) (the maximal concentration of agent achieved followingabsorption in, e.g., the bloodstream), T_(max) (the time required toachieve maximal concentration), T_(1/2) (the time required for theconcentration to fall by half), C_(min) (the minimal concentration ofagent following metabolism and excretion), AUC (area under the curve ofconcentration versus time, a measure of the overall amount ofbioavailability), concentrations in various tissues of interest(including, e.g., the rate of achieving desired concentrations, theoverall levels, and the duration of maintaining desired levels), and E.(the maximal effect achieved).

The methods of treatment provided herein include combination therapieswith a therapeutic agent for the treatment of a disease or disorder forwhich the therapeutic agent threats. Any therapeutic agent thatameliorates and or otherwise lessens the severity of a disease orcondition can be combined with a modified PH20 polypeptide providedherein in order to increase the bioavailability of such therapeuticagent. In particular, modified PH20 polypeptides provided herein can beused in each and all of the combinations described in applications seee.g., U.S. Publication Nos. US20040268425; US20050260186; US20060104968and U.S. Appl. Serial Nos. 12/381,844, published as U.S. Publication No.US20100074885; 12/386,249, published as U.S. Publication No.US20090311237; 12/387,225, published as U.S. Publication No.US20090304665; and Ser. No. 12/386,222, published as U.S. PublicationNo. US2010003238 in place of the disclosed hyaluronidase orhyaluronidase degrading enzyme.

Modified PH20 polypeptides can be administered prior to, subsequent to,intermittently with or simultaneously with the therapeutic agentpreparation. Generally, the modified PH20 polypeptide is administeredprior to or simultaneously with administration of the therapeutic agentpreparation to permit the PH20 to degrade the hyaluronic acid in theinterstitial space. The PH20 can be administered at a site differentfrom the site of administration of the therapeutic molecule or thesoluble PH20 can be administered at a site the same as the site ofadministration of the therapeutic molecule.

Examples of pharmaceutical, therapeutic and cosmetic agents andmolecules that can be administered with hyaluronidase include, but arenot limited to, a chemotherapeutic or anticancer agent, an analgesicagent, an antibiotic agent, an anti-inflammatory agent, an antimicrobialagent, an amoebicidal agent, a trichomonacidal agent, an anti-Parkinsonagent, an anti-malarial agent, an anticonvulsant agent, ananti-depressant agent, an anti-arthritic agent, an anti-fungal agent, anantihypertensive agent, an antipyretic agent, an anti-parasitic agent,an antihistamine agent, an alpha-adrenergic agonist agent, an alphablocker agent, an anesthetic agent, a bronchial dilator agent, a biocideagent, a bactericide agent, a bacteriostatic agent, a beta adrenergicblocker agent, a calcium channel blocker agent, a cardiovascular drugagent, a contraceptive agent, a cosmetic or esthetic agent, adecongestant agent, a diuretic agent, a depressant agent, a diagnosticagent, an electrolyte agent, a hypnotic agent, a hormone agent, ahyperglycemic agent, a muscle relaxant agent, a muscle contractantagent, an ophthalmic agent, a parasympathomimetic agent, a psychicenergizer agent, a sedative agent, a sleep inducer, a sympathomimeticagent, a tranquilizer agent, a urinary agent, a vaginal agent, aviricide agent, a vitamin agent, a non-steroidal anti-inflammatoryagent, or an angiotensin converting enzyme inhibitor agent, and anycombination thereof. In particular, therapeutic agents includeantibodies, including monoclonal antibodies, bisphosphonates, insulins,coagulation factors, cytokines and Immun Globulins.

For example, modified PH20 polypeptides provided herein can be used toincrease the delivery of chemotherapeutic agents. Hyaluronidases havealso been used to enhance the activity of chemotherapeutics and/or theaccessibility of tumors to chemotherapeutics (Schuller et al., 1991,Proc. Amer. Assoc. Cancer Res. 32:173, abstract no. 1034; Czejka et al.,1990, Pharmazie 45:H.9; Baumgartner et al., (1988) Reg. Cancer Treat.1:55-58; Zanker et al., (1986) Proc. Amer. Assoc. Cancer Res. 27:390).Combination chemotherapy with hyaluronidase is effective in thetreatment of a variety of cancers including urinary bladder cancer (Hornet al., (1985) J. Surg. Oncol. 28:304-307), squamous cell carcinoma(Kohno et al., (1994) J. Cancer Res. Oncol. 120:293-297), breast cancer(Beckenlehner et al., (1992) J. Cancer Res. Oncol. 118:591-596), andgastrointestinal cancer (Scheithauer et al., (1988) Anticancer Res.8:391-396). In this example, the modified PH20 hyaluronidase enhancespenetration of chemotherapeutic or other anti-cancer agents into solidtumors, thereby treating the disease.

Compositions containing soluble PH20 can be injected intratumorally withanti-cancer agents or intravenously for disseminated cancers or hard toreach tumors. The anticancer agent can be a chemotherapeutic, anantibody, a peptide, or a gene therapy vector, virus or DNA.Additionally, hyaluronidase can be used to recruit tumor cells into thecycling pool for sensitization in previously chemorefractory tumors thathave acquired multiple drug resistance (St Croix et al., (1998) CancerLett September 131(1): 35-44).

Exemplary anti-cancer agents that can be administered after, coincidentwith or before administration of a soluble PH20, such as an esPH20,include, but are not limited to Acivicins; Aclarubicins; Acodazoles;Acronines; Adozelesins; Aldesleukins; Alemtuzumabs; Alitretinoins(9-Cis-Retinoic Acids); Allopurinols; Altretamines; Alvocidibs;Ambazones; Ambomycins; Ametantrones; Amifostines; Aminoglutethimides;Amsacrines; Anastrozoles; Anaxirones; Ancitabines; Anthramycins;Apaziquones; Argimesnas; Arsenic Trioxides; Asparaginases; Asperlins;Atrimustines; Azacitidines; Azetepas; Azotomycins; Banoxantrones;Batabulins; Batimastats; BCG Live; Benaxibines; Bendamustines;Benzodepas; Bexarotenes; Bevacizumab; Bicalutamides; Bietaserpines;Biricodars; Bisantrenes; Bisantrenes; Bisnafide Dimesylates; Bizelesins;Bleomycins; Bortezomibs; Brequinars; Bropirimines; Budotitanes;Busulfans; Cactinomycins; Calusterones; Canertinibs; Capecitabines;Caracemides; Carbetimers; Carboplatins; Carboquones; Carmofurs;Carmustines with Polifeprosans; Carmustines; Carubicins; Carzelesins;Cedefingols; Celecoxibs; Cemadotins; Chlorambucils; Cioteronels;Ciplactin; Cirolemycins; Cisplatins; Cladribines; Clanfenurs;Clofarabines; Crisnatols; Cyclophosphamides; Cytarabine liposomals;Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas;Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins;Decitabines; Denileukin Diftitoxes; Dexniguldipines; Dexonas;Dexrazoxanes; Dezaguanines; Diaziquones; Dibrospidiums; Dienogests;Dinalins; Disermolides; Docetaxels; Dofequidars; Doxifluridines;Doxorubicin liposomals; Doxorubicin HCL; Doxorubicin HCL liposomeinjection; Doxorubicins; Droloxifenes; Dromostanolone Propionates;Duazomycins; Ecomustines; Edatrexates; Edotecarins; Eflornithines;Elacridars; Elinafides; Elliott's B Solutions; Elsamitrucins; Emitefurs;Enloplatins; Enpromates; Enzastaurins; Epipropidines; Epirubicins;Epoetin alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines;Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s;Etoposides; Etoprines; Exemestanes; Exisulinds; Fadrozoles; Fazarabines;Fenretinides; Filgrastims; Floxuridines; Fludarabines; Fluorouracils;5-fluorouracils; Fluoxymesterones; Flurocitabines; Fosquidones;Fostriecins; Fostriecins; Fotretamines; Fulvestrants; Galarubicins;Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins; Geroquinols;Gimatecans; Gimeracils; Gloxazones; Glufosfamides; Goserelin acetates;Hydroxyureas; Ibritumomabs/Tiuxetans; Idarubicins; Ifosfamides;Ilmofosines; Ilomastats; Imatinib mesylates; Imexons; Improsulfans;Indisulams; Inproquones; Interferon alfa-2as; Interferon alfa-2bs;Interferon Alfas; Interferon Betas; Interferon Gammas; Interferons;Interleukin-2s and other Interleukins (including recombinantInterleukins); Intoplicines; Iobenguanes [131-I]; Iproplatins;Irinotecans; Irsogladines; Ixabepilones; Ketotrexates; L-Alanosines;Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles; Leucovorins;Leuprolides; Leuprorelins (Leuprolides); Levamisoles; Lexacalcitols;Liarozoles; Lobaplatins; Lometrexols; Lomustines/CCNUs; Lomustines;Lonafarnibs; Losoxantrones; Lurtotecans; Mafosfamides; Mannosulfans;Marimastats; Masoprocols; Maytansines; Mechlorethamines;Mechlorethamines/Nitrogen mustards; Megestrol acetates; Megestrols;Melengestrols; Melphalans; Melphalan L-PAMs; Menogarils; Mepitiostanes;Mercaptopurines; 6-Mecaptopurine; Mesnas; Metesinds; Methotrexates;Methoxsalens; Metomidates; Metoprines; Meturedepas; Miboplatins;Miproxifenes; Misonidazoles; Mitindomides; Mitocarcins; Mitocromins;Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs;Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes;Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes;Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone Phenpropionates;Nedaplatins; Nelarabines; Nemorubicins; Nitracrines; Nocodazoles;Nofetumomabs; Nogalamycins; Nolatrexeds; Nortopixantrones; Octreotides;Oprelvekins; Ormaplatins; Ortataxels; Oteracils; Oxaliplatins;Oxisurans; Oxophenarsines; Paclitaxels; Pamidronates; Patupilones;Pegademases; Pegaspargases; Pegfilgrastims; Peldesines; Peliomycins;Pelitrexols; Pemetrexeds; Pentamustines; Pentostatins; Peplomycins;Perfosfamides; Perifosines; Picoplatins; Pinafides; Pipobromans;Piposulfans; Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds;Plicamycin Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimersodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines;Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins;Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans;Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins; Sabarubicins;Safingols; Sargramostims; Satraplatins; Sebriplatins; Semustines;Simtrazenes; Sizofirans; Sobuzoxanes; Sorafenibs; Sparfosates; SparfosicAcids; Sparsomycins; Spirogermaniums; Spiromustines; Spiroplatins;Spiroplatins; Squalamines; Streptonigrins; Streptovarycins;Streptozocins; Sufosfamides; Sulofenurs; Sunitinib Malate; 6-TG;Tacedinalines; Talcs; Talisomycins; Tallimustines; Tamoxifens;Tariquidars; Tauromustines; Tecogalans; Tegafurs; Teloxantrones;Temoporfins; Temozolomides; Teniposides/VM-26s; Teniposides;Teroxirones; Testolactones; Thiamiprines; Thioguanines; Thiotepas;Tiamiprines; Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics;Tirapazamines; Topixantrones; Topotecans; Toremifenes; Tositumomabs;Trabectedins (Ecteinascidin 743); Trastuzumabs; Trestolones;Tretinoins/ATRA; Triciribines; Trilostanes; Trimetrexates; TriplatinTetranitrates; Triptorelins; Trofosfamides; Tubulozoles; Ubenimexs;Uracil Mustards; Uredepas; Valrubicins; Valspodars; Vapreotides;Verteporfins; Vinblastines; Vincristines; Vindesines; Vinepidines;Vinflunines; Vinformides; Vinglycinates; Vinleucinols; Vinleurosines;Vinorelbines; Vinrosidines; Vintriptols; Vinzolidines; Vorozoles;Xanthomycin A's (Guamecyclines); Zeniplatins; Zilascorbs [2-H];Zinostatins; Zoledronate; Zorubicins; and Zosuquidars, for example:

Aldesleukins (e.g., PROLEUKIN®); Alemtuzumabs (e.g., CAMPATH®);Alitretinoins (e.g., PANRETIN®); Allopurinols (e.g., ZYLOPRIM®);Altretamines (e.g., HEXALEN®); Amifostines (e.g., ETHYOL®); Anastrozoles(e.g., ARIMIDEX®); Arsenic Trioxides (e.g., TRISENOX®); Asparaginases(e.g., ELSPAR®); BCG Live (e.g., TICE® BCG); Bexarotenes (e.g.,TARGRETIN®); Bevacizumab (AVASTIN®); Bleomycins (e.g., BLENOXANE R);Busulfan intravenous (e.g., BUSULFEX R); Busulfan orals (e.g.,MYLERAN™); Calusterones (e.g., METHOSARB®); Capecitabines (e.g.,XELODA®); Carboplatins (e.g., PARAPLATIN®); Carmustines (e.g., BCNU®,BiCNU®); Carmustines with Polifeprosans (e.g., GLIADEL® Wafer);Celecoxibs (e.g., CELEBREX®); Chlorambucils (e.g., LEUKERAN®));Cisplatins (e.g., PLATINOL®); Cladribines (e.g., LEUSTATIN®, 2-CdA®);Cyclophosphamides (e.g., CYTOXAN®, NEOSAR®); Cytarabines (e.g.,CYTOSAR-U®); Cytarabine liposomals (e.g., DepoCyt®); Dacarbazines (e.g.,DTIC-Domeu): Dactinomycins (e.g., COSMEGEN®); Darbepoetin Alfas (e.g.,ARANESP®); Daunorubicin liposomals (e. g. DAUNOXOME®);Daunorubicins/Daunomycins (e.g., CERUBIDINE®); Denileukin Diftitoxes(e.g., ONTAK®); Dexrazoxanes (e.g., ZINECARD®); Docetaxels (e.g.,TAXOTERE R); Doxorubicins (e.g., ADRIAMYCIN®, RUBEX®); Doxorubicinliposomals, including Doxorubicin HCL liposome injections (e.g., DOXILR); Dromostanolone propionates (e.g., DROMOSTANOLONE® and MASTERONE RInjection); Elliott's B Solutions (e.g., Elliott's B Solution®);Epirubicins (e.g., ELLENCE®); Epoetin alfas (e.g., EPOGEN®);Estramustines (e.g., EMCYT®); Etoposide phosphates (e.g., ETOPOPHOS®);Etoposide VP-16s (e.g., VEPESID®); Exemestanes (e.g., AROMASIN®);Filgrastims (e.g., NEUPOGEN®)); Floxuridines (e.g., FUDR®); Fludarabines(e.g., FLUDARA®); Fluorouracils incl. 5-FUs (e.g., ADRUCIL®);Fulvestrants (e.g., FASLODEX®); Gemcitabines (e.g., GEMZAR®);Gemtuzumabs/Ozogamicins (e.g., MYLOTARG®); Goserelin acetates (e.g.,ZOLADEX®); Hydroxyureas (e.g., HYDREA®); Ibritumomabs/Tiuxetans (e.g.,ZEVALIN®); Idarubicins (e.g., IDAMYCIN®); Ifosfamides (e.g., IFEX®);Imatinib mesylates (e.g., GLEEVEC®); Interferon alfa-2as (e.g.,ROFERON-A®); Interferon alfa-2bs (e.g., INTRON A®); Irinotecans (e.g.,CAMPTOSAR®); Letrozoles (e.g., FEMARA®); Leucovorins (e.g.,WELLCOVORIN®, LEUCOVORIN®); Levamisoles (e.g., ERGAMISOL®);Lomustines/CCNUs (e.g., CeeNU®); Mechlorethamines/Nitrogen mustards(e.g., MUSTARGEN®); Megestrol acetates (e.g., MEGACE®);Melphalans/L-PAMs (e.g., ALKERAN®); Mercaptopurine incl. 6-MPs (e.g.,PURINETHOL®); Mesnas (e.g., MESNEX®); Methotrexates; Methoxsalens (e.g.,UVADEX®); Mitomycin Cs (e.g., MUTAMYCINED, MITOZYTREX®); Mitotanes(e.g., LYSODREN®); Mitoxantrones (e.g., NOVANTRONE®); NandrolonePhenpropionates (e.g., DURABOLIN-50®); Nofetumomabs (e.g., VERLUMA®);Oprelvekins (e.g., NEUMEGA®); Oxaliplatins (e.g., ELOXATIN®);Paclitaxels (e.g., PAXENE®, TAXOL®); Pamidronates (e.g., AREDIA®);Pegademases (e.g., ADAGEN®); Pegaspargases (e.g., ONCASPAR®);Pegfilgrastims (e.g., NEULASTA®)); Pentostatins (e.g., NIPENT®);Pipobromans (e.g., VERCYTE®); Plicamycin/Mithramycins (e.g.,MITHRACIN®); Porfimer sodiums (e.g., PHOTOFRIN®); Procarbazines (e.g.,MATULANE®); Quinacrines (e.g., ATABRINE®); Rasburicases (e.g., ELITEK®);Rituximabs (e.g., RITUXAN®)); Sargramostims (e.g., PROKINE®);Streptozocins (e.g., ZANOSAR®); Sunitinib Malates (e.g., SUTENT®); Talcs(e.g., SCLEROSOL®); Tamoxifens (e.g., NOLVADEX®); Temozolomides (e.g.,TEMODAR®); Teniposides/VM-26s (e.g., VUMON®); Testolactones (e.g.,TESLAC®); Thioguanines incl. 6-TG; Thiotepas (e.g., THEOPLEX®);Topotecans (e.g., HYCAMTIN®); Toremifenes (e.g., FARESTON®);Tositumomabs (e.g., BEXXAR®); Trastuzumabs (e.g., HERCEPTIN®);Tretinoins/ATRA (e.g., VESANOID R); Uracil Mustards; Valrubicins (e.g.,VALSTAR0); Vinblastines (e.g., VELBAN®); Vincristines (e.g., ONCOVIN®);Vinorelbines (e.g., NAVELBINE®); and Zoledronates (e.g., ZOMETA®).

For example, exemplary antibiotic agents include, but are not limitedto, Aminoglycosides; Amphenicols; Ansamycins; Carbacephems; Carbapenems;Cephalosporins or Cephems; Cephamycins; Clavams; Cyclic lipopeptides;Diaminopyrimidines; Ketolides; Lincosamides; Macrolides; Monobactams;Nitrofurans; Oxacephems; Oxazolidinones; Penems, thienamycins andmiscellaneous beta-lactams; Penicillins; Polypeptides antibiotics;Quinolones; Sulfonamides; Sulfones; Tetracyclines; and other antibiotics(such as Clofoctols, Fusidic acids, Hexedines, Methenamines,Nitrofurantoins Nitroxolines, Ritipenems, Taurolidines, Xibomols).

Also included among exemplary therapeutic agents are coagulation factorsor other blood modifiers such as antihemophilic factors, anti-inhibitorcoagulant complexes, antithrombin III, coagulation Factor V, coagulationFactor VIII, coagulation Factor IX, plasma protein fractions, vonWillebrand factors; antiplatelet agents (including, for example,abciximabs, anagrelides, cilostazols, clopidogrel bisulfates,dipyridamoles, epoprostenols, eptifibatides, tirofibans; colonystimulating factors (CSFs) (including, for example, Granulocyte CSFs andGranulocyte Macrophage CSFs); erythropoiesis stimulators (including, forexample, erythropoietins such as darbepoetin alfas) and epoetin alfas;hemostatics and albumins (including, for example, aprotinins,combinations of antihemophilic factors and plasma, DesmopressinAcetates, and albumins); immune globulins, as well as hepatitis B immuneglobulins; thrombin inhibitors (including for example direct thrombininhibitors and lepirudin), and drotrecogin alfas; anticoagulants(including, for example, dalteparins, enoxaparins and other heparins,and warfarins).

Exemplary antibodies or other therapeutic agents include, but are notlimited to, Cetuximab (e.g., IMC-C225; Erbitux®); Trastuzumab (e.g.,Herceptin®); Rituximab (e.g., Rituxant; MabTherat); Bevacizumab (e.g.,Avastint); Alemtuzumab (e.g., Campatht; Campath-1H®; Mabcampath®);Panitumumab (e.g., ABX-EGF; Vectibix®); Ranibizumab (e.g., Lucentis®);Ibritumomab; Ibritumomab tiuxetan (e.g., Zevalin R); Tositumomab; IodineI 131 Tositumomab (e.g., BEXXAR®); Catumaxomab (e.g., Removab®);Gemtuzumab; Gemtuzumab ozogamicin (e.g., Mylotarg R); Abatacept (e.g.,CTLA4-Ig; Orencia®); Belatacept (L104EA29YIg; LEA29Y; LEA); Ipilimumab(e.g., MDX-010; MDX-101); Tremelimumab (e.g., ticilimumab; CP-675,206);PRS-010 (see e.g., US20090042785); PRS-050 (see e.g., U.S. Pat. No.7,585,940; US20090305982); Aflibercept (VEGF Trap, AVE005; Holash etal., (2002) PNAS 99:11393-11398); Volociximab (M200); F200 (Chimeric(human/murine) IgG4 Fab fragment of Volociximab (M200)); MORAb-009Mouse/human chimeric IgG1(US20050054048); Soluble fusionprotein:Anti-mesothelin Fv linked to a truncated Pseudomonas exotoxin A(SS1P (CAT-5001); US20070189962); Cixutumumab (IMC-A12); Nimotuzumab(h-R3) (Spicer (2005) Curr Opin Mol Ther 7:182-191); Zalutumumab(HuMax-EGFR; Lammerts van Bueren et al. (2008) PNAS 105:6109-14);Necitumumab IMC-11F8 (Li et al. (2008) Structure 16:216-227); Sym004(Pedersen et al. 2010 Cancer Res 70:588-597); and mAb-425.

In particular, therapeutic agents include, but are not limited to,immunoglobulins, Interferon beta, Interferon alpha-2as, Interferonalpha-1s, Interferon alpha-n3s, Interferon beta-1, Interferon beta-1as,Interferon gamma-1bs, Peg-interferon alpha-2 and Peginterferonalpha-2bs, insulin, a bisphosphate (e.g., Pamidronates or Zoledronates),Docetaxels, Doxorubicins, Doxorubicin liposomals and bevacizumabs.

Other exemplary therapeutic agents that can be combined byco-administration and/or co-formulation with a modified PH20 polypeptideprovided herein, include, but are not limited to, Adalimumabs,Agalsidase Betas, Alefacepts, Ampicillins, Anakinras, AntipoliomyeliticVaccines, Anti-Thymocytes, Azithromycins, Becaplermins, Caspofungins,Cefazolins, Cefepimes, Cefotetans, Ceftazidimes, Ceftriaxones,Cetuximabs, Cilastatins, Clavulanic Acids, Clindamycins, DarbepoetinAlfas, Daclizumabs, Diphtheria, Diphtheria antitoxins, DiphtheriaToxoids, Efalizumabs, Epinephrines, Erythropoietin Alphas, Etanercepts,Filgrastims, Fluconazoles, Follicle-Stimulating Hormones, FollitropinAlphas, Follitropin Betas, Fosphenytoins, Gadodiamides, Gadopentetates,Gatifloxacins, Glatiramers, GM-CSF's, Goserelins, Goserelin acetates,Granisetrons, Haemophilus Influenza B's, Haloperidols, Hepatitisvaccines, Hepatitis A Vaccines, Hepatitis B Vaccines, IbritumomabTiuxetans, Ibritumomabs, Tiuxetans, Immunoglobulins, Hemophilusinfluenza vaccines, Influenza Virus Vaccines, Infliximabs, Insulins,Insulin Glargines, Interferons, Interferon alphas, Interferon Betas,Interferon Gammas, Interferon alpha-2a's, Interferon alpha-2b's,Interferon alpha-1's, Interferon alpha-n3's, Interferon Betas,Interferon Beta-1a's, Interferon Gammas, Interferon alpha-consensus,Iodixanols, Iohexols, Iopamidols, Ioversols, Ketorolacs, Laronidases,Levofloxacins, Lidocaines, Linezolids, Lorazepams, Measles Vaccines,Measles virus, Mumps viruses, Measles-Mumps-Rubella Virus Vaccines,Rubella vaccines, Medroxyprogesterones, Meropenems, Methylprednisolones,Midazolams, Morphines, Octreotides, Omalizumabs, Ondansetrons,Palivizumabs, Pantoprazoles, Pegaspargases, Pegfilgrastims,Peg-Interferon Alfa-2a's, Peg-Interferon Alfa-2b's, Pegvisomants,Pertussis vaccines, Piperacillins, Pneumococcal Vaccines andPneumococcal Conjugate Vaccines, Promethazines, Reteplases, Somatropins,Sulbactams, Sumatriptans, Tazobactams, Tenecteplases, Tetanus PurifiedToxoids, Ticarcillins, Tositumomabs, Triamcinolones, TriamcinoloneAcetonides, Triamcinolone hexacetonides, Vancomycins, Varicella Zosterimmunoglobulins, Varicella vaccines, other vaccines, Alemtuzumabs,Alitretinoins, Allopurinols, Altretamines, Amifostines, Anastrozoles,Arsenics, Arsenic Trioxides, Asparaginases, Bacillus Calmette-Guerin(BCG) vaccines, BCG Live, Bexarotenes, Bleomycins, Busulfans, Busulfanintravenous, Busulfan orals, Calusterones, Capecitabines, Carboplatins,Carmustines, Carmustines with Polifeprosans, Celecoxibs, Chlorambucils,Cisplatins, Cladribines, Cyclophosphamides, Cytarabines, Cytarabineliposomals, Dacarbazines, Dactinomycins, Daunorubicin liposomals,Daunorubicins, Daunomycins, Denileukin Diftitoxes, Dexrazoxanes,Docetaxels, Doxorubicins, Doxorubicin liposomals, Dromostanolonepropionates, Elliott's B Solutions, Epirubicins, Epoetin alfas,Estramustines, Etoposides, Etoposide phosphates, Etoposide VP-16s,Exemestanes, Floxuridines, Fludarabines, Fluorouracils, 5-Fluorouracils,Fulvestrants, Gemcitabines, Gemtuzumabs, Ozogamicins, Gemtuzumabozogamicins, Hydroxyureas, Idarubicins, Ifosfamides, Imatinib mesylates,Irinotecans, Letrozoles, Leucovorins, Levamisoles, Lomustines, CCNUs,Mechlorethamines, Nitrogen mustards, Megestrols, Megestrol acetates,Melphalans, L-PAMs, Mercaptopurines, 6-Mercaptopurines, Mesnas,Methotrexates, Methoxsalens, Mitomycins, Mitomycin C's, Mitotanes,Mitoxantrones, Nandrolones, Nandrolone Phenpropionates, Nofetumomabs,Oprelvekins, Oxaliplatins, Paclitaxels, Pamidronates, Pegademases,Pentostatins, Pipobromans, Plicamycins, Mithramycins, Porfimers,Porfimer sodiums, Procarbazines, Quinacrines, Rasburicases, Rituximabs,Sargramostims, Streptozocins, Talcs, Tamoxifens, Temozolomides,Teniposides, Testolactones, Thioguanines, 6-Thioguanines,Triethylenethiophosphoramides (Thiotepas), Topotecans, Toremifenes,Trastuzumabs, Tretinoins, Uracil Mustards, Valrubicins, Vinblastines,Vincristines, Vinorelbines, Zoledronates, Acivicins, Aclarubicins,Acodazoles, Acronines, Adozelesins, Aldesleukins, Retinoic Acids,Alitretinoins, 9-Cis-Retinoic Acids, Alvocidibs, Ambazones, Ambomycins,Ametantrones, Aminoglutethimides, Amsacrines, Anaxirones, Ancitabines,Anthramycins, Apaziquones, Argimesnas, Asperlins, Atrimustines,Azacitidines, Azetepas, Azotomycins, Banoxantrones, Batabulins,Batimastats, Benaxibines, Bendamustines, Benzodepas, Bicalutamides,Bietaserpines, Biricodars, Bisantrenes, Bisnafide Dimesylates,Bizelesins, Bortezomibs, Brequinars, Bropirimines, Budotitanes,Cactinomycins, Canertinibs, Caracemides, Carbetimers, Carboquones,Carmofurs, Carubicins, Carzelesins, Cedefingols, Cemadotins,Chlorambucils, Cioteronels, Cirolemycins, Clanfenurs, Clofarabines,Crisnatols, Decitabines, Dexniguldipines, Dexormaplatins, Dezaguanines,Diaziquones, Dibrospidiums, Dienogests, Dinalins, Disermolides,Dofequidars, Doxifluridines, Droloxifenes, Duazomycins, Ecomustines,Edatrexates, Edotecarins, Eflomithines, Elacridars, Elinafides,Elsamitrucins, Emitefurs, Enloplatins, Enpromates, Enzastaurins,Epipropidines, Eptaloprosts, Erbulozoles, Esorubicins, Etanidazoles,Etoglucids, Etoprines, Exisulinds, Fadrozoles, Fazarabines,Fenretinides, Fluoxymesterones, Flurocitabines, Fosquidones,Fostriecins, Fotretamines, Galarubicins, Galocitabines, Geroquinols,Gimatecans, Gimeracils, Gloxazones, Glufosfamides, Ilmofosines,Ilomastats, Imexons, Improsulfans, Indisulams, Inproquones,Interleukins, Interleukin-2s, recombinant Interleukins, Intoplicines,Iobenguanes, Iproplatins, Irsogladines, Ixabepilones, Ketotrexates,L-Alanosines, Lanreotides, Lapatinibs, Ledoxantrones, Leuprolides,Leuprorelins, Lexacalcitols, Liarozoles, Lobaplatins, Lometrexols,Lonafarnibs, Losoxantrones, Lurtotecans, Mafosfamides, Mannosulfans,Marimastats, Masoprocols, Maytansines, Mechlorethamines, Melengestrols,Melphalans, Menogarils, Mepitiostanes, Metesinds, Metomidates,Metoprines, Meturedepas, Miboplatins, Miproxifenes, Misonidazoles,Mitindomides, Mitocarcins, Mitocromins, Mitoflaxones, Mitogillins,Mitoguazones, Mitomalcins, Mitonafides, Mitoquidones, Mitospers,Mitozolomides, Mivobulins, Mizoribines, Mofarotenes, Mopidamols,Mubritinibs, Mycophenolic Acids, Nedaplatins, Neizarabines,Nemorubicins, Nitracrines, Nocodazoles, Nogalamycins, Nolatrexeds,Nortopixantrones, Ormaplatins, Ortataxels, Oteracils, Oxisurans,Oxophenarsines, Patupilones, Peldesines, Peliomycins, Pelitrexols,Pemetrexeds, Pentamustines, Peplomycins, Perfosfamides, Perifosines,Picoplatins, Pinafides, Piposulfans, Pirfenidones, Piroxantrones,Pixantrones, Plevitrexeds, Plomestanes, Porfiromycins, Prednimustines,Propamidines, Prospidiums, Pumitepas, Puromycins, Pyrazofurins,Ranimustines, Riboprines, Ritrosulfans, Rogletimides, Roquinimexs,Rufocromomycins, Sabarubicins, Safingols, Satraplatins, Sebriplatins,Semustines, Simtrazenes, Sizofirans, Sobuzoxanes, Sorafenibs,Sparfosates, Sparfosic Acids, Sparsomycins, Spirogermaniums,Spiromustines, Spiroplatins, Squalamines, Streptonigrins,Streptovarycins, Sufosfamides, Sulofenurs, Tacedinalines, Talisomycins,Tallimustines, Tariquidars, Tauromustines, Tecogalans, Tegafurs,Teloxantrones, Temoporfins, Teroxirones, Thiamiprines, Tiamiprines,Tiazofurins, Tilomisoles, Tilorones, Timcodars, Timonacics,Tirapazamines, Topixantrones, Trabectedins, Ecteinascidin 743,Trestolones, Triciribines, Trilostanes, Trimetrexates, TriplatinTetranitrates, Triptorelins, Trofosfamides, Tubulozoles, Ubenimexs,Uredepas, Valspodars, Vapreotides, Verteporfins, Vinblastines,Vindesines, Vinepidines, Vinflunines, Vinformides, Vinglycinates,Vinleucinols, Vinleurosines, Vinrosidines, Vintriptols, Vinzolidines,Vorozoles, Xanthomycin A's, Guamecyclines, Zeniplatins, Zilascorbs[2-H], Zinostatins, Zorubicins, Zosuquidars, Acetazolamides, Acyclovirs,Adipiodones, Alatrofloxacins, Alfentanils, Allergenic extracts, Alpha1-proteinase inhibitors, Alprostadils, Amikacins, Amino acids,Aminocaproic acids, Aminophyllines, Amitriptylines, Amobarbitals,Amrinones, Analgesics, Anti-poliomyelitic vaccines, Anti-rabic serums,Anti-tetanus immunoglobulins, tetanus vaccines, Antithrombin IIIs,Antivenom serums, Argatrobans, Arginines, Ascorbic acids, Atenolols,Atracuriums, Atropines, Aurothioglucoses, Azathioprines, Aztreonams,Bacitracins, Baclofens, Basiliximabs, Benzoic acids, Benztropines,Betamethasones, Biotins, Bivalirudins, Botulism antitoxins, Bretyliums,Bumetanides, Bupivacaines, Buprenorphines, Butorphanols, Calcitonins,Calcitriols, Calciums, Capreomycins, Carboprosts, Carnitines,Cefamandoles, Cefoperazones, Cefotaximes, Cefoxitins, Ceftizoximes,Cefuroximes, Chloramphenicols, Chloroprocaines, Chloroquines,Chlorothiazides, Chlorpromazines, Chondroitinsulfuric acids,Choriogonadotropin alfas, Chromiums, Cidofovirs, Cimetidines,Ciprofloxacins, Cisatracuriums, Clonidines, Codeines, Colchicines,Colistins, Collagens, Corticorelin ovine triflutates, Corticotrophins,Cosyntropins, Cyanocobalamins, Cyclosporines, Cysteines, Dacliximabs,Dalfopristins, Dalteparins, Danaparoids, Dantrolenes, Deferoxamines,Desmopressins, Dexamethasones, Dexmedetomidines, Dexpanthenols,Dextrans, Iron dextrans, Diatrizoic acids, Diazepams, Diazoxides,Dicyclomines, Digibinds, Digoxins, Dihydroergotamines, Diltiazems,Diphenhydramines, Dipyridamoles, Dobutamines, Dopamines, Doxacuriums,Doxaprams, Doxercalciferols, Doxycyclines, Droperidols, Dyphyllines,Edetic acids, Edrophoniums, Enalaprilats, Ephedrines, Epoprostenols,Ergocalciferols, Ergonovines, Ertapenems, Erythromycins, Esmolols,Estradiols, Estrogenics, Ethacrynic acids, Ethanolamines, Ethanols,Ethiodized oils, Etidronic acids, Etomidates, Factor VIIIs, Famotidines,Fenoldopams, Fentanyls, Flumazenils, Fluoresceins, Fluphenazines, Folicacids, Fomepizoles, Fomivirsens, Fondaparinuxs, Foscarnets,Fosphenytoins, Furosemides, Gadoteridols, Gadoversetamides,Ganciclovirs, Gentamicins, Glucagons, Glucoses, Glycines,Glycopyrrolates, Gonadorelins, Gonadotropin chorionics, Haemophilus Bpolysaccharides, Hemins, Herbals, Histamines, Hydralazines,Hydrocortisones, Hydromorphones, Hydroxocobalamins, Hydroxyzines,Hyoscyamines, Ibutilides, Imiglucerases, Indigo carmines, Indomethacins,Iodides, Iopromides, lothalamic acids, loxaglic acids, Ioxilans,Isoniazids, Isoproterenols, Japanese encephalitis vaccines, Kanamycins,Ketamines, Labetalols, Lepirudins, Levobupivacaines, Levothyroxines,Lincomycins, Liothyronines, Luteinizing hormones, Lyme disease vaccines,Mangafodipirs, Manthtols, Meningococcal polysaccharide vaccines,Meperidines, Mepivacaines, Mesoridazines, Metaraminols, Methadones,Methocarbamols, Methohexitals, Methyldopates, Methylergonovines,Metoclopramides, Metoprolols, Metronidazoles, Minocyclines, Mivacuriums,Morrhuic acids, Moxifloxacins, Muromonab-CD3s, Mycophenolate mofetils,Nafcillins, Nalbuphines, Nalmefenes, Naloxones, Neostigmines,Niacinamides, Nicardipines, Nitroglycerins, Nitroprussides,Norepinephrines, Orphenadrines, Oxacillins, Oxymorphones,Oxytetracyclines, Oxytocins, Pancuroniums, Panthenols, Pantothenicacids, Papaverines, Peginterferon-alpha (e.g., interferon alpha 2a or2b), Penicillin Gs, Pentamidines, Pentazocines, Pentobarbitals,Perflutrens, Perphenazines, Phenobarbitals, Phentolamines,Phenylephrines, Phenytoins, Physostigmines, Phytonadiones, Polymyxin bs,Pralidoximes, Prilocaines, Procainamides, Procaines, Prochlorperazines,Progesterones, Propranolols, Pyridostigmine hydroxides, Pyridoxines,Quinidines, Quinupristins, Rabies immunoglobulins, Rabies vaccines,Ranitidines, Remifentanils, Riboflavins, Rifampins, Ropivacaines,Samariums, Scopolamines, Seleniums, Sermorelins, Sincalides, Somatrems,Spectinomycins, Streptokinases, Streptomycins, Succinylcholines,Sufentanils, Sulfamethoxazoles, Tacrolimuses, Terbutalines,Teriparatides, Testosterones, Tetanus antitoxins, Tetracaines,Tetradecyl sulfates, Theophyllines, Thiamines, Thiethylperazines,Thiopentals, Thyroid stimulating hormones, Tinzaparins, Tirofibans,Tobramycins, Tolazolines, Tolbutamides, Torsemides, Tranexamic acids,Treprostinils, Trifluoperazines, Trimethobenzamides, Trimethoprims,Tromethamines, Tuberculins, Typhoid vaccines, Urofollitropins,Urokinases, Valproic acids, Vasopressins, Vecuroniums, Verapamils,Voriconazoles, Warfarins, Yellow fever vaccines, Zidovudines, Zincs,Ziprasidone hydrochlorides, Aclacinomycins, Actinomycins, Adriamycins,Azaserines, 6-Azauridines, Carzinophilins, Chromomycins, Denopterins,6-Diazo-5-Oxo-L-Norleucines, Enocitabines, Floxuridines, Olivomycins,Pirarubicins, Piritrexims, Pteropterins, Tegafurs, Tubercidins,Alteplases, Arcitumomabs, bevacizumabs, Botulinum Toxin Type A's,Botulinum Toxin Type B's, Capromab Pendetides, Daclizumabs, Dornasealfas, Drotrecogin alfas, Imciromab Pentetates, and Iodine-131's.

Delivery of Insulin

Methods provided herein include methods of co-administering a modifiedPH20 polypeptide and an insulin to increase subcutaneous delivery of theinsulin, such as a fast-acting insulin (see e.g., U.S. Pat. Nos.7,767,429; 7,846,431; U.S. Publication No. US20090304665; and U.S.application Ser. Nos. 13/507,263; 13/507,262 and 13/507,261). Suchmethods include methods of direct administration, and pump andcontinuous infusion methods, including open and closed pump systems. Forexample, exemplary insulins that can be administered with a modifiedPH20 hyaluronidase provided herein are fast-acting insulins or insulinanalogs. For example, a co-administered insulin includes a regularinsulin, insulin aspart, insulin lispro, insulin glulisine or othersimilar analog variants. Exemplary insulins are insulins that contain anA chain set forth in SEQ ID NO:862 and a B chain set forth in SEQ IDNO:863 or variants that contain one or more amino acid modificationscompared to a human insulin set forth in SEQ ID NO: 862 and 863 (A and Bchains) For example, exemplary insulin analogs are known to one of skillin the art, and include, but are not limited to, those set forth in SEQID NO:862 (A-chain) and having a B-chain set forth in any of SEQ ID NOs:865-867.

The co-formulations can be administered subcutaneously to treat anycondition that is amenable to treatment with insulin. Therapeutic usesinclude, but are not limited to, treatment for type 1 diabetes mellitus,type 2 diabetes mellitus, gestational diabetes, and for glycemic controlin critically ill patients. For example, the co-formulations of a fastacting insulin and hyaluronan degrading enzyme can be administeredsubcutaneously in discrete doses, such as via a syringe or insulin pen,prior to a meal as prandial insulin therapy in subjects with diabetes toachieve glycemic control. The co-formulations also can be administeredsubcutaneously or intraperitoneally using an insulin pump or in thecontext of a closed loop system to continuously control blood glucoselevels throughout the day and night and/or to control post-prandialglycemic excursions. It is within the skill of a treating physician toidentify such diseases or conditions.

For any disease or condition, including all those exemplified above, forwhich a fast-acting insulin is indicated or has been used and for whichother agents and treatments are available, the co-formulations can beused in combination therewith. Depending on the disease or condition tobe treated, exemplary combinations include, but are not limited to,combinations with anti-diabetic drugs, including, but not limited to,sulfonylureas, biguanides, meglitinides, thiazolidinediones,alpha-glucosidase inhibitors, peptide analogs, including glucagon-likepeptide (GLP) analogs and, gastric inhibitory peptide (GIP) analogs andDPP-4 inhibitors. In another example, the co-formulations of a fastacting insulin and modified PH20 polypeptide described herein can beadministered in combination with, prior to, intermittently with, orsubsequent to, one or more other insulins, including fast-actinginsulin, and basal-acting insulins.

2. Methods of Hyaluronan-Associated Diseases and Conditions (e.g.,Tumors)

In particular, PH20 hyaluronidase can be used to treathyaluronan-associated diseases or conditions. Typically,hyaluronan-associated diseases and conditions are associated withelevated hyaluronan expression in a tissue, cell, or body fluid (e.g.,tumor tissue or tumor-associated tissue, blood, or interstitial space)compared to a control, e.g., another tissue, cell or body fluid. Theelevated hyaluronan expression can be elevated compared to a normaltissue, cell or body fluid, for example, a tissue, cell or body fluidthat is analogous to the sample being tested, but isolated from adifferent subject, such as a subject that is normal (i.e., does not havea disease or condition, or does not have the type of disease orcondition that the subject being tested has), for example, a subjectthat does not have a hyaluronan-associated disease or condition. Theelevated hyaluronan expression can be elevated compared to an analogoustissue from another subject that has a similar disease or condition, butwhose disease is not as severe and/or is not hyaluronan-associated orexpresses relatively less hyaluronan and thus is hyaluronan-associatedto a lesser degree. For example, the subject being tested can be asubject with a hyaluronan-associated cancer, where the HA amounts in thetissue, cell or fluid are relatively elevated compared to a subjecthaving a less severe cancer, such as an early stage, differentiated orother type of cancer. In another example, the cell, tissue or fluidcontains elevated levels of hyaluronan compared to a control sample,such as a fluid, tissue, extract (e.g., cellular or nuclear extract),nucleic acid or peptide preparation, cell line, biopsy, standard orother sample, with a known amount or relative amount of HA, such as asample, for example a tumor cell line, known to express relatively lowlevels of HA, such as exemplary tumor cell lines described herein thatexpress low levels of HA, for example, the HCT 116 cell line, the HT29cell line, the NCI H460 cell line, the DU145 cell line, the Capan-1 cellline, and tumors from tumor models generated using such cell lines.

Hyaluronan-associated diseases and conditions include those associatedwith high interstitial fluid pressure, such as disc pressure,proliferative disorders, such as cancer and benign prostatichyperplasia, and edema. Edema can result from or be manifested in, forexample, organ transplant, stroke or brain trauma. Proliferativedisorders include, but are not limited to, cancer, smooth muscle cellproliferation, systemic sclerosis, cirrhosis of the liver, adultrespiratory distress syndrome, idiopathic cardiomyopathy, lupuserythematosus, retinopathy, e.g., diabetic retinopathy or otherretinopathies, cardiac hyperplasia, reproductive system associateddisorders, such as benign prostatic hyperplasia (BPH) and ovarian cysts,pulmonary fibrosis, endometriosis, fibromatosis, hamartomas,lymphangiomatosis, sarcoidosis, desmoid tumors. Cancers include solidand lymphatic/blood tumors and metastatic disease, and undifferentiatedtumors. The tumors amenable to treatment typically exhibit cellularand/or stromal expression of a hyaluronan, compared to a non-canceroustissue of the same tissue type or compared to a non-metastatic tumor ofthe same tumor-type. Cancers include any one or more of ovarian cancer,in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer,pancreatic cancer, other gastric cancers, non-small cell lung cancer,breast cancer, brain cancer and colon cancer.

Modified PH20 polypeptides provided herein, such as PEGylated formsthereof, can be used to treat tumors. Thus, in addition to its indirectanticancer effects, hyaluronidases also have direct anticarcinogeniceffects. Hyaluronidase prevents growth of tumors transplanted into mice(De Maeyer et al., (1992) Int. J. Cancer 51:657-660) and inhibits tumorformation upon exposure to carcinogens (Pawlowski et al., (1979) Int. J.Cancer 23:105-109; Haberman et al., (1981) Proceedings of the 17thAnnual Meeting of the American Society of Clinical Oncology, Washington,D.C., 22:105, abstract no. 415). PH20 hyaluronidase has been shown totreat various tumors (see e.g., U.S. Publication No. US2010/0003238 andU.S. application Ser. No. 13/135,817, published as U.S. Publication No.US20120020951).

The hyaluronan-rich cancer can be a cancer in which the cancer cellsproduce HALOS, cancers that have elevated expression of hyaluronan (asdetermined by immunostaining, e.g., histological staining of sectionsfrom the tumor), cancers that have elevated HAS2 (Hyaluronan synthase2), cancers that do not produce hyaluronidase (HYAL1) in vitro.Hyaluronan-rich cancers can be identified by any method for assessinghyaluronan expression, and other known methods for assaying protein/mRNAexpression.

Several hyaluronan-rich cancers have been identified. In some cases,hyaluronan expression correlates with poor prognosis, for example,decreased survival rate and/or recurrence-free survival rate,metastases, angiogenesis, cancer cell invasion into other tissues/areas,and other indicators of poor prognosis. Such correlation has beenobserved, for example, in hyaluronan-rich tumors including ovariancancer, SCC, ISC, prostate cancer, lung cancer, including non-small-celllung cancer (NSCLC), breast cancer, colon cancer and pancreatic cancer(see, for example, Anttila et al., Cancer Research, 60:150-155 (2000);Karvinen et al., British Journal of Dermatology, 148:86-94 (2003);Lipponen et al., Eur. Journal of Cancer, 849-856 (2001); Pirinen et al.,Int. J. Cancer: 95: 12-17 (2001); Auvinen et al., American Journal ofPathology, 156(2):529-536 (2000); Ropponen et al., Cancer Research, 58:342-347 (1998)). Thus, hyaluronan-rich cancers can be treated byadministration of a hyaluronidase, such as a soluble PH20, to treat oneor more symptoms of the cancer. Hyaluronan-rich tumors include, but arenot limited to those of the prostate, breast, colon, ovarian, stomach,head and neck and other tumors and cancers.

Other hyaluronan-associated diseases or conditions that are associatedwith excess glycosaminoglycans and that can be treated with a modifiedPH20 polypeptide provided herein include, but are not limited to,cardiovascular disease (e.g., following ischemia reperfusion; inarteriosclerosis); vitrectomy and ophthalmic disorders and conditions(e.g., in methods to liquefy the vitreous humor of the eye; reducepostoperative pressure; other ocular surgical procedures such asglaucoma, vitreous and retina surgery and in corneal transplantation);in hypodermoclysis (i.e., infusion of fluids and electrolytes into thehypodermis of the skin); cosmetic applications (e.g., in the treatmentof cellulite, “pigskin” edema or “orange peel” edema); organtransplantation (e.g., associated with interstitial edemas in connectionwith grafting of an organ); pulmonary disease.

3. Other Uses

In further examples of its therapeutic use, modified PH20 polypeptidesprovided herein, can be used for such purposes as an antidote to localnecrosis from paravenous injection of necrotic substances such as vincaalkaloids (Few et al. (1987) Amer. J. Matern. Child Nurs. 12, 23-26),treatment of ganglion cysts (Paul et al. (1997) J Hand Surg. 22 (2):219-21) and treatment of tissue necrosis due to venous insufficiency(Elder et al. (1980) Lancet 648-649). Modified PH20 polypeptides alsocan be used to treat ganglion cysts (also known as a wrist cyst, Biblecyst, or dorsal tendon cyst), which are the most common soft tissue massof the hand and are fluid filled sacs that can be felt below the skin.

Modified PH20 polypeptides can be used in the treatment of spinal cordinjury by degrading chondroitin sulfate proteoglycans (CSPGs). Followingspinal cord injury, glial scars containing CSPGs are produced byastrocytes. CSPGs play a crucial role in the inhibition of axon growth.In addition, the expression of CSPG has been shown to increase followinginjury of the central nervous system (CNS). Soluble PH20 also can beutilized for the treatment of herniated disks in a process known aschemonucleolysis. Chondroitinase ABC, an enzyme cleaving similarsubstrates as hyaluronidase, can induce the reduction of intradiscalpressure in the lumbar spine. There are three types of disk injuries. Aprotruded disk is one that is intact but bulging. In an extruded disk,the fibrous wrapper has torn and the NP has oozed out, but is stillconnected to the disk. In a sequestered disk, a fragment of the NP hasbroken loose from the disk and is free in the spinal canal.Chemonucleolysis is typically effective on protruded and extruded disks,but not on sequestered disk injuries.

4. Contraception

Modified PH20 polypeptides provided herein can be used as vaccines incontraceptive applications. PH20 is present in the male reproductivetract, and is expressed in both the testis and epididymis and is presentin sperm. PH20 plays a role in fertilization by facilitating entry ofthe sperm through the cumulus layer surrounding the unfertilized egg.PH20 also is able to bind to hyaluronic acid (HA) on the zona pellucidaduring early phases of fertilization. This binding also initiatesintracellular signaling that aids in the acrosome reaction. Immunizationwith PH20 has been shown to be an effective contraceptive in male guineapigs (Primakoff et al. (1988) Nature 335:543-546, Tung et al. (1997)Biol. Reprod. 56:1133-1141). It also has been shown to be an effectivecontraceptive in female guinea pigs due to the generation of anti-PH20antibodies that prevent sperm and egg binding. In examples herein, themodified PH20 polypeptides can be inactive enzymes, such as anydescribed in Sections C.2. The polypeptides can be administered directlyor can be administered as a recombinant virus to deliver the antigen.

I. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1

Generation of Recombinant Human pH20 Hyaluronidase (rHuPH20)A. Generation of a Soluble rHuPH20-Expressing Cell Line

A recombinant human PH20 hyaluronidase designated rHuPH20 was generatedas described in published U.S. Publication No. US20110053247. Briefly,the pCI-PH20-IRES-DHFR-SV40pa (HZ24) plasmid (set forth in SEQ ID NO:5)was used to transfect Chinese Hamster Ovary (CHO cells) (see e.g., U.S.Pat. Nos. 7,767,429 and 7,781,607 and U.S. Publication No.2006-0104968). The HZ24 plasmid vector for expression of soluble rHuPH20contains a pCI vector backbone (Promega), DNA encoding amino acids 1-482of human PH20 hyaluronidase (SEQ ID NO:2), an internal ribosomal entrysite (IRES) from the ECMV virus (Clontech), and the mouse dihydrofolatereductase (DHFR) gene. The pCI vector backbone also includes DNAencoding the Beta-lactamase resistance gene (AmpR), an f1 origin ofreplication, a Cytomegalovirus immediate-early enhancer/promoter region(CMV), a chimeric intron, and an SV40 late polyadenylation signal(SV40). The DNA encoding the soluble rHuPH20 construct contains an NheIsite and a Kozak consensus sequence prior to the DNA encoding themethionine at amino acid position 1 of the native 35 amino acid signalsequence of human PH20, and a stop codon following the DNA encoding thetyrosine corresponding to amino acid position 482 of the human PH20hyaluronidase set forth in SEQ ID NO:2, followed by a BamHI restrictionsite.

Non-transfected DG44 CHO cells growing in GIBCO Modified CD-CHO mediafor DHFR(−) cells, supplemented with 4 mM Glutamine and 18 mL/LPlurionic F68/L (Gibco), were seeded at 0.5×10⁶ cells/mL in a shakerflask in preparation for transfection. Cells were grown at 37° C. in 5%CO₂ in a humidified incubator, shaking at 120 rpm. Exponentially growingnon-transfected DG44 CHO cells were tested for viability prior totransfection.

Sixty million viable cells of the non-transfected DG44 CHO cell culturewere pelleted and resuspended to a density of 2×10⁷ cells in 0.7 mL of2× transfection buffer (2× HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mMKCl, 1.4 mM Na₂HPO₄, 12 mM dextrose). To each aliquot of resuspendedcells, 0.09 mL (250 μg) of the linear HZ24 plasmid (linearized byovernight digestion with Cla I (New England Biolabs) was added, and thecell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics)electroporation cuvettes at room temperature. A negative controlelectroporation was performed with no plasmid DNA mixed with the cells.The cell/plasmid mixes were electroporated with a capacitor discharge of330 V and 960 μF or at 350 V and 960 μF.

The cells were removed from the cuvettes after electroporation andtransferred into 5 mL of Modified CD-CHO media for DHFR(−) cells,supplemented with 4 mM Glutamine and 18 mL/L Plurionic F68/L (Gibco),and allowed to grow in a well of a 6-well tissue culture plate withoutselection for 2 days at 37° C. in 5% CO₂ in a humidified incubator.

Two days post-electroporation, 0.5 mL of tissue culture media wasremoved from each well and tested for the presence of hyaluronidaseactivity, using the microturbidity assay described in Example 8. Theresults are set forth in Table 6.

TABLE 6 Initial Hyaluronidase Activity of HZ24 Transfected DG44 CHOcells at 40 hours post-transfection Activity Dilution (Units/mL)Transfection 1 330 V 1 to 10 0.25 Transfection 2 350 V 1 to 10 0.52Negative Control 1 to 10 0.015

Cells from Transfection 2 (350V) were collected from the tissue culturewell, counted and diluted to 1×10⁴ to 2×10⁴ viable cells per mL. A 0.1mL aliquot of the cell suspension was transferred to each well of five,96 well round bottom tissue culture plates. One hundred microliters ofCD-CHO media (GIBCO) containing 4 mM GlutaMAX™-1 supplement (GIBCO™,Invitrogen Corporation) and without hypoxanthine and thymidinesupplements were added to the wells containing cells (final volume 0.2mL). Ten clones were identified from the 5 plates grown withoutmethotrexate (Table 7).

TABLE 7 Hyaluronidase activity of identified clones Plate/Well IDRelative Hyaluronidase 1C3 261 2C2 261 3D3 261 3E5 243 3C6 174 2G8 1031B9 304 2D9 273 4D10 302

Six HZ24 clones were expanded in culture and transferred into shakerflasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E11, and4D10 were plated into 96-well round bottom tissue culture plates using atwo-dimensional infinite dilution strategy in which cells were diluted1:2 down the plate, and 1:3 across the plate, starting at 5000 cells inthe top left hand well. Diluted clones were grown in a background of 500non-transfected DG44 CHO cells per well, to provide necessary growthfactors for the initial days in culture. Ten plates were made persubclone, with 5 plates containing 50 nM methotrexate and 5 plateswithout methotrexate.

Clone 3D3 produced 24 visual subclones (13 from the no methotrexatetreatment, and 11 from the 50 nM methotrexate treatment). Significanthyaluronidase activity was measured in the supernatants from 8 of the 24subclones (>50 Units/mL), and these 8 subclones were expanded into T-25tissue culture flasks. Clones isolated from the methotrexate treatmentprotocol were expanded in the presence of 50 nM methotrexate. Clone3D35M was further expanded in 500 nM methotrexate giving rise to clonesproducing hyaluronidase activity in excess of 1,000 Units/mL in shakerflasks (clone 3D35M; or Gen′ 3D35M). A master cell bank (MCB) of the3D35M cells was then prepared.

B. Production Gen2 Cells Containing Soluble human PH20 (rHuPH20)

The Gen′ 3D35M cell line described in Example 1.A was adapted to highermethotrexate levels to produce generation 2 (Gen2) clones. 3D35M cellswere seeded from established methotrexate-containing cultures into CDCHO medium containing 4 mM GlutaMAX-1™ and 1.0 μM methotrexate. Thecells were adapted to a higher methotrexate level by growing andpassaging them 9 times over a period of 46 days in a 37° C., 7% CO₂humidified incubator. The amplified population of cells was cloned outby limiting dilution in 96-well tissue culture plates containing mediumwith 2.0 μM methotrexate. After approximately 4 weeks, clones wereidentified and clone 3E10B was selected for expansion. 3E10B cells weregrown in CD CHO medium containing 4 mM GlutaMAX-1™ and 2.0 μMmethotrexate for 20 passages. A master cell bank (MCB) of the 3E10B cellline was created and frozen and used for subsequent studies.

Amplification of the cell line continued by culturing 3E10B cells in CDCHO medium containing 4 mM GlutaMAX-1™ and 4.0 μM methotrexate. Afterthe 12th passage, cells were frozen in vials as a research cell bank(RCB). One vial of the RCB was thawed and cultured in medium containing8.0 μM methotrexate. After 5 days, the methotrexate concentration in themedium was increased to 16.0 μM, then 20.0 μM 18 days later. Cells fromthe 8th passage in medium containing 20.0 μM methotrexate were clonedout by limiting dilution in 96-well tissue culture plates containing CDCHO medium containing 4 mM GlutaMAX-1™ and 20.0 μM methotrexate. Cloneswere identified 5-6 weeks later and clone 2B2 was selected for expansionin medium containing 20.0 μM methotrexate. After the 11th passage, 2B2cells were frozen in vials as a research cell bank (RCB).

The resultant 2B2 cells are dihydrofolate reductase deficient (dhfr-)DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20).The soluble PH20 is present in 2B2 cells at a copy number ofapproximately 206 copies/cell. Southern blot analysis of Spe I-, Xba I-and BamH I/Hind III-digested genomic 2B2 cell DNA using arHuPH20-specific probe revealed the following restriction digestprofile: one major hybridizing band of 18 7.7 kb and four minorhybridizing bands (˜13.9, ˜6.6, ˜5.7 and ˜4.6 kb) with DNA digested withSpe I; one major hybridizing band of ˜5.0 kb and two minor hybridizingbands (˜13.9 and ˜6.5 kb) with DNA digested with Xba I; and one singlehybridizing band of ˜1.4 kb observed using 2B2 DNA digested with BamHI/Hind III.

C. Production of Gen2 soluble rHuPH20 in 300 L Bioreactor Cell Culture

A vial of HZ24-2B2 was thawed and expanded from shaker flasks through 36L spinner flasks in CD-CHO media (Invitrogen, Carlsbad, Calif.)supplemented with 20 μM methotrexate and GlutaMAX-1™ (Invitrogen).Briefly, the vial of cells was thawed in a 37° C. water bath, medium wasadded and the cells were centrifuged. The cells were re-suspended in a125 mL shake flask with 20 mL of fresh medium and placed in a 37° C., 7%CO₂ incubator. The cells were expanded up to 40 mL in the 125 mL shakeflask. When the cell density reached greater than 1.5×10⁶ cells/mL, theculture was expanded into a 125 mL spinner flask in a 100 mL culturevolume. The flask was incubated at 37° C., 7% CO₂. When the cell densityreached greater than 1.5×10⁶ cells/mL, the culture was expanded into a250 mL spinner flask in 200 mL culture volume, and the flask wasincubated at 37° C., 7% CO₂. When the cell density reached greater than1.5×10⁶ cells/mL, the culture was expanded into a 1 L spinner flask in800 mL culture volume and incubated at 37° C., 7% CO₂. When the celldensity reached greater than 1.5×10⁶ cells/mL the culture was expandedinto a 6 L spinner flask in 5000 mL culture volume and incubated at 37°C., 7% CO₂. When the cell density reached greater than 1.5×10⁶ cells/mLthe culture was expanded into a 36 L spinner flask in 32 L culturevolume and incubated at 37° C., 7% CO₂.

A 400 L reactor was sterilized and 230 mL of CD-CHO media were added.Before use, the reactor was checked for contamination. Approximately 30L cells were transferred from the 36 L spinner flasks to the 400 Lbioreactor (Braun) at an inoculation density of 4.0×10⁵ viable cells permL and a total volume of 260 L. Parameters were: temperature setpoint,37° C.; Impeller Speed 40-55 RPM; Vessel Pressure: 3 psi; Air Sparge0.5-1.5 L/Min.; Air Overlay: 3 L/min. The reactor was sampled daily forcell counts, pH verification, media analysis, protein production andretention. Also, during the run nutrient feeds were added. At 120 hrs(day 5), 10.4L of Feed #1 Medium (4×CD-CHO+33 g/L Glucose+160 mL/LGlutamax-1™+83 mL/L Yeastolate+33 mg/L rHulnsulin) was added. At 168hours (day 7), 10.8 L of Feed #2 (2×CD-CHO+33 g/L Glucose+80 mL/LGlutamax-1™+167 mL/L Yeastolate+0.92 g/L Sodium Butyrate) was added, andculture temperature was changed to 36.5° C. At 216 hours (day 9), 10.8 Lof Feed #3 (1× CD-CHO+50 g/L Glucose+50 mL/L Glutamax-1™+250 mL/LYeastolate+1.80 g/L Sodium Butyrate) was added, and culture temperaturewas changed to 36° C. At 264 hours (day 11), 10.8 L of Feed #4(1×CD-CHO+33 g/L Glucose+33 mL/L Glutamax-1™+250 mL/L Yeastolate+0.92g/L Sodium Butyrate) was added, and culture temperature was changed to35.5° C. The addition of the feed media was observed to dramaticallyenhance the production of soluble rHuPH20 in the final stages ofproduction. The reactor was harvested at 14 or 15 days or when theviability of the cells dropped below 40%. The process resulted in afinal productivity of 17,000 Units per mL with a maximal cell density of12 million cells/mL. At harvest, the culture was sampled for mycoplasma,bioburden, endotoxin and virus in vitro and in vivo, by TransmissionElectron Microscopy (TEM) and enzyme activity.

The culture was pumped by a peristaltic pump through four Millistakfiltration system modules (Millipore) in parallel, each containing alayer of diatomaceous earth graded to 4-8 μm and a layer of diatomaceousearth graded to 1.4-1.1 μm, followed by a cellulose membrane, thenthrough a second single Millistak filtration system (Millipore)containing a layer of diatomaceous earth graded to 0.4-0.11 μm and alayer of diatomaceous earth graded to <0.1 μm, followed by a cellulosemembrane, and then through a 0.22 μm final filter into a sterile singleuse flexible bag with a 350 L capacity. The harvested cell culture fluidwas supplemented with 10 mM EDTA and 10 mM Tris to a pH of 7.5. Theculture was concentrated 10× with a tangential flow filtration (TFF)apparatus using four Sartoslice TFF 30 kDa molecular weight cut-off(MWCO) polyether sulfone (PES) filter (Sartorious), followed by a 10×buffer exchange with 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 into a 0.22 μmfinal filter into a 50 L sterile storage bag.

The concentrated, diafiltered harvest was inactivated for virus. Priorto viral inactivation, a solution of 10% Triton® X-100 detergent, and 3%tri (n-butyl) phosphate (TNBP) was prepared. The concentrated,diafiltered harvest was exposed to 1% Triton® X-100 detergent, and 0.3%TNBP for 1 hour in a 36 L glass reaction vessel immediately prior topurification on the Q column.

D. Purification of Gen2 soluble rHuPH20

A Q Sepharose (Pharmacia) ion exchange column (9 L resin, H=29 cm, D=20cm) was prepared. Wash samples were collected for a determination of pH,conductivity and endotoxin (LAL assay). The column was equilibrated with5 column volumes of 10 mM Tris, 20 mM Na₂SO₄, pH 7.5. Following viralinactivation, the concentrated, diafiltered harvest was loaded onto theQ column at a flow rate of 100 cm/hr. The column was washed with 5column volumes of 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 and 10 mM Hepes, 50mM NaCl, pH7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCl, pH7.0 into a 0.22 μm final filter into sterile bag. The eluate sample wastested for bioburden, protein concentration and hyaluronidase activity.A₂₈₀ absorbance readings were taken at the beginning and end of theexchange.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography wasnext performed. A Phenyl-Sepharose (PS) column (19-21 L resin, H=29 cm,D=30 cm) was prepared. The wash was collected and sampled for pH,conductivity and endotoxin (LAL assay). The column was equilibrated with5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate,and 0.1 mM CaCl₂, pH 7.0. The protein eluate from the Q sepharose columnwas supplemented with 2M ammonium sulfate, 1 M potassium phosphate and 1M CaCl₂ stock solutions to yield final concentrations of 5 mM, 0.5 M and0.1 mM, respectively. The protein was loaded onto the PS column at aflow rate of 100 cm/hr and the column flow thru collected. The columnwas washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1mM CaCl₂ pH 7.0 at 100 cm/hr and the wash was added to the collectedflow thru. Combined with the column wash, the flow through was passedthrough a 0.22 μm final filter into a sterile bag. The flow through wassampled for bioburden, protein concentration and enzyme activity.

An aminophenyl boronate column (Prometics) was prepared. The wash wascollected and sampled for pH, conductivity and endotoxin (LAL assay).The column was equilibrated with 5 column volumes of 5 mM potassiumphosphate, 0.5 M ammonium sulfate. The PS flow through containingpurified protein was loaded onto the aminophenyl boronate column at aflow rate of 100 cm/hr. The column was washed with 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20mM bicine, 0.5 M ammonium sulfate, pH 9.0. The column was washed with 20mM bicine, 100 mM sodium chloride, pH 9.0. The protein was eluted with50 mM Hepes, 100 mM NaCl, pH 6.9 and passed through a sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The hydroxyapatite (HAP) column (Biorad) was prepared. The wash wascollected and tested for pH, conductivity and endotoxin (LAL assay). Thecolumn was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, 0.1mM CaCl₂, pH 7.0. The aminophenyl boronate purified protein wassupplemented to final concentrations of 5 mM potassium phosphate and 0.1mM CaCl₂ and loaded onto the HAP column at a flow rate of 100 cm/hr. Thecolumn was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1mM CaCl₂. The column was next washed with 10 mM potassium phosphate, pH7, 100 mM NaCl, 0.1 mM CaCl₂. The protein was eluted with 70 mMpotassium phosphate, pH 7.0 and passed through a 0.22 μm sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The HAP purified protein was then passed through a virus removal filter.The sterilized Viosart filter (Sartorius) was first prepared by washingwith 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filteredbuffer was sampled for pH and conductivity. The HAP purified protein waspumped via a peristaltic pump through the 20 nM virus removal filter.The filtered protein in 70 mM potassium phosphate, pH 7.0 was passedthrough a 0.22 μm final filter into a sterile bag. The filtered samplewas tested for protein concentration, enzyme activity, oligosaccharide,monosaccharide and sialic acid profiling. The sample also was tested forprocess related impurities.

The protein in the filtrate was then concentrated to 10 mg/mL using a 10kDa molecular weight cut off (MWCO) Sartocon Slice tangential flowfiltration (TFF) system (Sartorius). The filter was first prepared bywashing with 10 mM histidine, 130 mM NaCl, pH 6.0 and the permeate wassampled for pH and conductivity. Following concentration, theconcentrated protein was sampled and tested for protein concentrationand enzyme activity. A 6× buffer exchange was performed on theconcentrated protein into the final buffer: 10 mM histidine, 130 mMNaCl, pH 6.0. Following buffer exchange, the concentrated protein waspassed though a 0.22 μm filter into a 20 L sterile storage bag. Theprotein was sampled and tested for protein concentration, enzymeactivity, free sulfydryl groups, oligosaccharide profiling andosmolality. Lot number WRS2 was used as a standard in the assaysdescribed below, the results showed that the test description forappearance was clear and colorless; the pH was 7.4; the endotoxin levelwas <0.01 EU/mL; the osmolality was 308 mOsm/Kg; the density was 1.005g/mL; the rHuPH20 content was 1.3 ppm; and the hyaluronidase activitywas 145 USP U/mL.

The sterile filtered bulk protein was then asceptically dispensed at 20mL into 30 mL sterile Teflon vials (Nalgene). The vials were then flashfrozen and stored at −20±5° C.

Example 2 Generation of PH20 Mutant Library

A. Cloning and Mutagenesis

In this example, a human hyaluronidase PH20 library was created bycloning DNA encoding human PH20 into a plasmid followed by transfectionand protein expression.

The library was created by mutagenesis of a PH20 template that is acodon optimized version of PH20 with an Ig Kappa leader sequence.Specifically, for generating the library of variants, theHZ24-PH20(OHO)-IRES-SEAP expression vector (set forth in SEQ ID NO:4)was used as a template, which contains the sequence of nucleotidesencoding PH20 set forth in SEQ ID NO:1, which encodes a precursor PH20set forth in SEQ ID NO:2 or a mature PH20 set forth in SEQ ID NO:3lacking residues 1-22 corresponding to the IgK signal sequence. Thebackbone of the vector was derived from the original HZ24 vectorcontaining the DHFR selection marker (see Example 1 and SEQ ID NO:5)with the addition of an IgK leader sequence and codon optimization. Theexpression vector also was modified to contain the gene for secretedalkaline phosphatase (SEAP). Hence, in addition to sequence encodingPH20, the HZ24-PH20(OHO)-IRES-SEAP expression vector also contains aninternal ribosome entry site (EMCV IRES) that is linked to the codingsequence for the gene for secreted alkaline phosphatase (SEAP), and asingle CMV promoter that drives expression of PH20 and SEAP in theconstruct. It also contains a gene for ampilcillin resistance. Withreference to the sequence of nucleotides set forth in SEQ ID NO:4, thesequence of nucleotides encoding PH20 corresponds to nucleotides1058-2464 (including the IgK leader sequence), the sequence ofnucleotides encoding SEAP corresponds to nucleotides 2970-4529, and theampicillin resistance gene corresponds to nucleotides 5778-6635.

The first library was made to generate encoded variant proteins whereineach of residues 23-469 of SEQ ID NO:2 (corresponding to residues 1-447of SEQ ID NO:3 or residues 36-482 of SEQ ID NO:6) was changed to one ofabout 15 amino acid residues, such that each member contained a singleamino change. The resulting library contained 6753 variant members, eachcontaining a single amino acid mutation compared to residues 23-469 ofSEQ ID NO:2 (corresponding to residues 1-447 of SEQ ID NO:3 or residues36-482 of SEQ ID NO:6). Glycerol stocks of the resulting library wereprepared and stored at −80° C. The amino acid replacements (mut) in eachmember are listed in Table 8 below, and correspond to amino acidreplacements with reference to the sequence of amino acids of PH20 setforth in SEQ ID NO:3 (and SEQ ID NOs: 7 or 32-66, which are the maturesequence of PH20 or other C-terminally truncated fragments thereof). Thecorresponding mutated codons (cod) of each PH20 variant in the libraryare also listed in Table 8, and correspond to nucleotide residue changesin the corresponding encoding nucleotide for PH20 set forth as 1058-2464of SEQ ID NO:4. Each member was expressed and screened for hyaluoridaseactivity as described below.

TABLE 8 PH20 Variants mut cod mut cod mut cod mut cod mut cod mut codL001A GCG Y066S AGT R132N AAT G198T ACT V265G GGT I331K AAG L001C TGTY066T ACG R132P CCT G198V GTT V265H CAT I331L CTG L001D GAT Y066V GTGR132Q CAG G198W TGG V265I ATT I331Q CAG L001E GAG I067C TGT R132S AGTG198Y TAT V265K AAG I331R CGT L001F TTT I067D GAT R132T ACT Y199A GCGV265L CTG I331S AGT L001G GGT I067E GAG R132V GTG Y199C TGT V265M ATGI331T ACT L001H CAT I067F TTT R132Y TAT Y199E GAG V265N AAT I331W TGGL001K AAG I067G GGG S133A GCT Y199G GGG V265P CCT I331Y TAT L001N AATI067H CAT S133D GAT Y199H CAT V265Q CAG I332A GCT L001P CCG I067L TTGS133E GAG Y199I ATT V265R AGG I332C TGT L001Q CAG I067N AAT S133F TTTY199K AAG V265S TCT I332D GAT L001R CGG I067P CCG S133G GGG Y199L CTTV265W TGG I332E GAG L001S TCT I067Q CAG S133H CAT Y199N AAT V265Y TATI332F TTT L001T ACG I067R CGG S133I ATT Y199P CCT F266A GCG I332G GGTL001V GTG I067T ACG S133L CTG Y199Q CAG F266C TGT I332H CAT L001W TGGI067V GTT S133M ATG Y199R AGG F266D GAT I332K AAG N002A GCT I067W TGGS133N AAT Y199S TCG F266G GGG I332L CTG N002C TGT I067Y TAT S133P CCTY199T ACG F266H CAT I332N AAT N002F TTT D068A GCT S133R CGG Y199W TGGF266L CTT I332P CCT N002G GGG D068C TGT S133T ACT N200A GCT F266M CCGI332R AGG N002H CAT D068E GAG S133V GTT N200D GAT F266P ATG I332S AGTN002I ATT D068G GGG S133W TGG N200F CAG F266Q CAG I332T ACT N002K AAGD068H CAC I134A GCT N200G GGT F266R CGG I332Y TAT N002L TTG D068I ATTI134C TGT N200H CAT F266S TCG N333A GCT N002P CCG D068K AAG I134D GATN200K AAG F266T ACG N333E GAG N002Q CAG D068L TTG I134F TTT N200L CTGF266V GTG N333G GGT N002S AGT D068P CCT I134G GGG N200M ATG F266W TGGN333H CAT N002T ACG D068Q CAG I134H CAT N200P CCT F266Y TAT N333I ATTN002V GTT D068R CGG I134K AAG N200Q CAG A267D GAT N333K AAG N002W TGGD068S TCG I134L TTG N200R AGG A267E GAG N333L CTG N002Y TAT D068T ACTI134P CCT N200S TCT A267G GGT N333M ATG F003A GCT D068V GTG I134Q CAGN200T ACT A267H CAT N333P CCT F003E GAG D068Y TAT I134R CGT N200V GTGA267I ATT N333R CGG F003G GGG S069A GCT I134S TCG N200W TGG A267K AAGN333S AGT F003H CAT S069C TGT I134T ACT N200Y TAT A267L CTT N333T ACTF003I ATT S069E GAG I134V GTG G201A GCG A267M ATG N333V GTT F003K AAGS069F TTT I134W TGG G201E GAG A267N AAT N333W TGG F003L TTG S069G GGGE135A GCT G201F TTT A267P CCG N333Y TAT F003M ATG S069I ATT E135C TGTG201H CAT A267R AGG V334A GCT F003N AAT S069L CTT E135D GAT G201K AAGA267S TCT V334C TGT F003P CCT S069M ATG E135F TTT G201L CTT A267T GTGV334D GAT F003R CGT S069N AAT E135G GGG G201M ATG A267V ACT V334E GAGF003S TCG S069P CCT E135H CAT G201N AAT A267W TGG V334G GGG F003T ACTS069R CGT E135K AAG G201P CCT Y268A GCT V334H CAT F003V GTG S069T ACGE135L TTG G201Q CAG Y268C TGT V334L TTG F003Y TAT S069V GTT E135N AATG201R CGT Y268F TTT V334M ATG R004A GCG S069W TGG E135P CCT G201S TCGY268G GGG V334N AAT R004D GAT S069Y TAT E135Q CAG G201T ACG Y268H CATV334P CCT R004E GAG I070A GCT E135R CGG G201V GTG Y268K AAG V334Q CAGR004F TTT I070C TGT E135S TCT G201W TGG Y268L CTT V334R AGG R004G GGGI070F TTT E135W TGG S202A GCG Y268N AAT V334S TCT R004I ATT I070G GGGE135Y TAT S202E GAG Y268P CCT V334T ACT R004L TTG I070H CAT L136A GCTS202F TTT Y268Q CAG V334Y TAT R004M ATG I070K AAG L136C TGT S202G GGTY268R CGT T335A GCT R004N AAT I070L TTG L136D GAT S202H CAT Y268S TCGT335C TGT R004P CCT I070N AAT L136F TTT S202K AAG Y268T ACT T335F TTTR004S TCT I070P CCG L136G GGT S202M ATG Y268V GTG T335G GGT R004T ACGI070Q CAG L136H CAT S202N AAT Y268W TGG T335H CAT R004V GTG I070R CGTL136I ATT S202P CCT T269A GCT T335I ATT R004W TGG I070S TCT L136M ATGS202Q CAG T269C TGT T335K AAG R004Y TAT I070T ACT L136N AAT S202R CGTT269D GAT T335L TTG A005D GAT I070V GTT L136P CCT S202T ACG T269E GAGT335N AAT A005G GGG I070Y TAT L136Q CAG S202V GTT T269G GGT T335P CCTA005H CAT T071A GCT L136R CGT S202W TGG T269K AAG T335Q CAG A005I ATTT071C TGT L136S TCG S202Y TAT T269L CTG T335S TCT A005L CTT T071D GATL136T ACT C203A GCG T269M ATG T335V GTG A005M ATG T071E GAG L136W TGGC203D GAT T269N AAT T335W TGG A005N AAT T071G GGG V137A GCT C203E GAGT269P CCG T335Y TAT A005P CCG T071H CAT V137C TGT C203G GGG T269Q CAGL336A GCT A005Q CAG T071L TTG V137E GAG C203H CAT T269R AGG L336E GAGA005R AGG T071M ATG V137F TTT C203L CTT T269S TCG L336F TTT A005S TCGT071N AAT V137G GGG C203M ATG T269V GTG L336G GGG A005T ACG T071P CCTV137H CAT C203N AAT T269Y TAT L336H CAT A005V GTG T071Q CAG V137I ATTC203P CCG R270A GCT L336K AAG A005W TGG T071R CGG V137L TTG C203Q CAGR270C TGT L336M ATG A005Y TAT T071S TCG V137N AAT C203R AGG R270D GATL336N AAT P006A GCG T071V GTG V137P CCT C203S AGT R270E GAG L336P CCTP006D GAT T071Y TAT V137Q CAG C203T ACT R270F TTT L336R AGG P006E GAGG072A GCT V137R CGT C203V GTG R270G GGG L336S TCT P006F TTT G072C TGTV137S TCT C203W TGG R270H CAT L336T ACT P006G GGG G072D GAT V137T ACTF204A GCG R270I ATT L336V GTG P006H CAT G072E GAG V137W TGG F204C TGTR270M ATG L336W TGG P006K AAG G072F TTT V137Y TAT F204E GAG R270N AATL336Y TAT P006L CTT G072H CAT Q138A GCT F204G GGG R270P CCT A337C TGTP006N AAT G072I ATT Q138C TGT F204H CAT R270Q CAG A337F TTT P006Q CAGG072K AAG Q138E GAG F204I ATT R270S TCG A337G GGG P006R AGG G072L TTGQ138F TTT F204K AAG R270T ACT A337H CAT P006S AGT G072M ATG Q138G GGGF204L CTT R270V GTG A337I ATT P006T ACG G072P CCT Q138H CAT F204M ATGR270Y TAT A337K AAG P006V GTG G072Q CAG Q138I ATT F204P CCT I271A GCTA337L TTG P006W TGG G072R CGG Q138L TTG F204Q CAG I271D GAT A337M ATGP006Y TAT G072S TCT Q138M ATG F204R AGG I271E GAG A337N AAT P007A GCTG072T ACT Q138N AAT F204S AGT I271F TTT A337P CCT P007C TGT G072V GTGQ138R CGT F204T ACT I271G GGG A337R CGG P007D GAT G072W TGG Q138S AGTF204V GTG I271H CAT A337S TCT P007F TTT G072Y TAT Q138V GTT F204W TGGI271K AAG A337T ACT P007G GGT V073A GCG Q138W TGG N205A GCG I271L CTTA337V GTT P007H CAT V073C TGT Q138Y TAT N205D GAT I271M ATG A337W TGGP007I ATT V073D GAT Q139A GCT N205E GAG I271P CCT A338C TGT P007K AAGV073F TTT Q139C TGT N205F TTT I271R AGG A338D GAT P007L TTG V073G GGGQ139D GAT N205G GGG I271S AGT A338E GAG P007M ATG V073H CAT Q139E GAGN205K AAG I271T ACT A338F TTT P007Q CAG V073K AAG Q139F TTT N205L CTGI271V GTT A338G GGG P007R CGG V073L CTT Q139G GGG N205M ATG I271W TGGA338H CAT P007S AGT V073M ATG Q139H CAT N205P CCT V272A GCT A338I ATTP007T ACT V073P CCG Q139K AAG N205R AGG V272C TGT A338K AAG P007V GTGV073Q CAG Q139L CTG N205S TCG V272D GAT A338L CTT P007W TGG V073R TGGQ139M ATG N205T ACG V272E GAG A338P CCT P007Y TAT V073S TCG Q139P CCTN205V GTG V272G GGG A338Q CAG V008A GCT V073T ACG Q139R CGT N205W TGGV272H CAT A338R CGT V008D GAT V073W CGG Q139S TCT N205Y TAT V272K AAGA338S TCG V008E GAG T074A GCT Q139T ACT V206C TGT V272L TTG A338T ACTV008G GGT T074C TGT Q139V GTG V206D GAT V272M ATG A338V GTG V008H CATT074E GAG Q140A GCT V206F TTT V272N AAT K339D GAT V008I ATT T074F TTTQ140C TGT V206G GGG V272P CCT K339E GAG V008L TTG T074G GGT Q140D GATV206H CAT V272R AGG K339F TTT V008M ATG T074H CAT Q140F TTT V206I ATTV272S TCG K339G GGG V008N AAT T074K AAG Q140G GGG V206K AAG V272T ACTK339H CAT V008P CCT T074L TTG Q140H CAT V206L CTT V272W TGG K339L CTGV008Q CAG T074M ATG Q140I ATT V206M ATG F273A GCT K339M ATG V008R CGGT074N AAT Q140K AAG V206P CCG F273C TGT K339N AAT V008S TCT T074P CCGQ140L TTG V206Q CAG F273D GAT K339P CCT V008T ACT T074R CGG Q140M ATGV206R CGG F273G GGG K339R CGG V008W TGG T074S TCG Q140R CGG V206S TCTF273H CAT K339S AGT I009A GCT T074V GTG Q140S AGT V206T ACG F273I ATTK339T ACT I009C TGT T074W TGG Q140V GTG V206Y TAT F273L CTG K339V GTTI009D GAT V075A GCG Q140W TGG E207A GCT F273P CCT K339W TGG I009E GAGV075C TGT Q140Y TAT E207F TTT F273Q CAG K339Y TAT I009G GGG V075D GATN141A GCT E207G GGG F273R CGG M340A GCT I009H CAT V075F TTT N141D GATE207H CAT F273S TCG M340C TGT I009K AAG V075G GGG N141E GAG E207I ATTF273T ACG M340D GAT I009L CTT V075H CAT N141F TTT E207K AAG F273V GTTM340E GAG I009N AAT V075L CTT N141G GGT E207L TTG F273W TGG M340F TTTI009P CCT V075M ATG N141H CAT E207M ATG F273Y TAT M340G GGG I009Q CAGV075N AAT N141L TTG E207P CCG T274A GCG M340H CAT I009R CGG V075P CCGN141M ATG E207Q CAG T274C TGT M340K AAG I009S AGT V075Q CAG N141P CCTE207R AGG T274E GAG M340L CTG I009T ACG V075R CGT N141Q CAG E207S TCTT274F ATG M340P CCT I009V GTT V075S TCT N141R CGT E207T ACG T274G GGGM340R CGG P010D GAT V075T ACT N141S TCT E207V GTT T274H CAT M340S TCGP010E GAG V075W TGG N141T ACT E207W TGG T274L CTG M340T ACT P010F TTTV075Y TAT N141V GTT I208A GCT T274N AAT M340V GTG P010G GGT N076A GCTN141W TGG I208C TGT T274P CCT M340W TGG P010H CAT N076C TGT N141Y TATI208D GAT T274Q CAG C341A GCT P010I ATT N076D GAT V142C TGT I208E GAGT274R CGT C341E GAG P010L CTT N076F TTT V142D GAT I208G GGG T274S AGTC341G GGG P010M ATG N076G GGG V142E GAG I208K AAG T274V GTT C341H CATP010N AAT N076I ATT V142G GGG I208L TTG T274W TGG C341K AAG P010Q CAGN076K AAG V142H CAT I208M ATG T274Y TAT C341L TTG P010R CGG N076L CTGV142I ATT I208P CCG D275A GCT C341M ATG P010S TCG N076P CCT V142K AAGI208Q CAG D275C TGT C341N AAT P010T ACT N076Q CAG V142L TTG I208R CGTD275E GAG C341Q CAG P010W TGG N076R CGT V142M ATG I208S AGT D275F TTTC341R AGG P010Y TAT N076S AGT V142N AAT I208T ACG D275G GGG C341S TCTN011A GCG N076T ACT V142P CCT I208V GTG D275I ATT C341T ACT N011C TGTN076V GTT V142Q CAG I208W TGG D275K AAG C341V GTT N011D GAT N076W TGGV142R CGG K209A GCG D275L CTT C341W TGG N011E GAG G077D GAT V142S AGTK209C TGT D275M ATG C341Y TAT N011F TTT G077E GAG V142T ACT K209D GATD275Q CAG S342A GCT N011G GGG G077F TTT Q143C TGT K209E GAG D275R CGTS342D GAT N011H CAT G077H CAT Q143E GAG K209F TTT D275S TCG S342E GAGN011I ATT G077K AAG Q143F TTT K209G GGT D275T ACT S342F TTT N011K AAGG077L TTG Q143G GGG K209L CTG D275V GTG S342G GGG N011L CTG G077M ATGQ143H CAT K209N AAT D275W TGG S342H CAT N011P CCG G077N AAT Q143I ATTK209P CCG Q276C TGT S342I ATT N011S TCG G077P CCG Q143K AAG K209R CGGQ276D GAT S342K AAG N011T ACG G077Q CAG Q143L TTG K209S AGT Q276E GAGS342L TTG N011W TGG G077R CGT Q143M ATG K209T ACT Q276F TTT S342M ATGN011Y TAT G077S TCG Q143N AAT K209V GTT Q276G GGG S342P CCT V012A GCTG077T ACG Q143P CCT K209W TGG Q276H CAT S342Q CAG V012D GAT G077V GTGQ143R CGG K209Y TAT Q276I ATT S342R CGG V012E GAG G077Y TAT Q143S TCGR210A GCG Q276L CTT S342T ACT V012G GGG G078A GCG Q143T ACT R210C TGTQ276M ATG S342Y TAT V012H CAT G078C TGT Q143V GTG R210D GAT Q276P CCTQ343C TGT V012I ATT G078D GAT Q143Y TAT R210E GAG Q276R CGT Q343D GATV012K AAG G078H CAT L144A GCT R210G GGT Q276S AGT Q343E GAG V012L CTTG078I ATT L144E GAG R210K AAG Q276V GTT Q343F TTT V012M ATG G078K AAGL144F TTT R210L CTG Q276W TGG Q343G GGG V012N AAT G078L TTG L144G GGGR210M ATG Q276Y TAT Q343I ATT V012P CCG G078M ATG L144I ATT R210N AATV277A GCT Q343L CTT V012R AGG G078P CCG L144K AAG R210P CCT V277C TGTQ343M ATG V012S TCG G078Q CAG L144N AAT R210S TCG V277D GAT Q343P CCTV012T ACT G078R AGG L144P CCT R210T ACT V277E GAG Q343R AGG V012W TGGG078S TCG L144Q CAG R210V GTG V277G GGG Q343S AGT P013A GCT G078T ACTL144R CGT R210W TGG V277H CAT Q343T ACT P013E GAG G078V GTG L144S TCTR210Y TAT V277K AAG Q343V GTG P013F TTT G078Y TAT L144T ACT N211A GCGV277L TTG Q343W TGG P013G GGG I079A GCT L144V GTT N211C TGT V277M ATGQ343Y TAT P013H CAT I079D GAT L144W TGG N211F TTT V277N AAT V344E GAGP013I ATT I079F TTT L144Y TAT N211G GGG V277Q CAG V344F TTT P013L CTTI079G GGG S145A GCT N211H CAT V277R AGG V344G GGG P013M ATG I079H CATS145C TGT N211I ATT V277S TCT V344H CAT P013Q CAG I079K AAG S145D GATN211K AAG V277T ACT V344I ATT P013R CGT I079L TTG S145E GAG N211L CTGV277Y TAT V344L CTG P013S TCG I079N AAT S145F TTT N211M ATG L278A GCTV344M ATG P013T ACT I079P CCG S145G GGG N211P CCT L278E GAG V344N AATP013V GTG I079R CGT S145H CAT N211R CGG L278F TTT V344P CCT P013W TGGI079S AGT S145L TTG N211S AGT L278G GGG V344Q CAG P013Y TAT I079T ACTS145M ATG N211T ACT L278H CAT V344R CGT F014A GCG I079V GTT S145N AATN211V GTT L278I ATT V344S TCG F014D GAT I079W TGG S145P CCT N211W TGGL278K AAG V344T ACT F014E GAG I079Y TAT S145R CGT D212A GCT L278M TTTV344W TGG F014G GGT P080A GCG S145T ACT D212E GAG L278N AAT V344Y TATF014H CAT P080D GAT S145V GTT D212G GGG L278P CCG L345A GCT F014I ATTP080E GAG S145W TGG D212H CAT L278R CGT L345C TGT F014K AAG P080F TTTL146A GCT D212I ATT L278S TCT L345D GAT F014M ATG P080G GGG L146C TGTD212K AAG L278T ACT L345E GAG F014N AAT P080I ATT L146E GAG D212L CTGL278V GTT L345G GGG F014P CCT P080K AAG L146G GGG D212M ATG L278Y TATL345H CAT F014Q CAG P080L CTT L146H CAT D212N AAT K279A GCG L345K AAGF014R CGG P080M ATG L146I ATT D212P CCT K279C TGT L345N AAT F014T ACTP080N AAT L146K AAG D212Q CAG K279D GAT L345P CCT F014V GTG P080R AGGL146N AAT D212S TCG K279F TTT L345Q CAG F014W TGG P080S TCT L146P CCTD212T ACT K279G GGG L345R CGT L015A GCG P080T ACG L146Q CAG D212V GTGK279H CAT L345T ACT L015E GAG P080V GTG L146R CGG D212W TGG K279L CTGL345V GTT L015F TTT P080Y TAT L146S TCG D213A GCT K279P CCT L345W TGGL015G GGG Q081A GCT L146T ACT D213E GAG K279Q CAG L345Y TAT L015K AAGQ081C TGT L146V GTT D213G GGG K279R AGG C346A GCT L015M ATG Q081E GAGL146Y TAT D213H CAT K279S TCT C346D GAT L015N AAT Q081F TTT T147A GCTD213K AAG K279T ACG C346F TTT L015P CCG Q081G GGG T147C TGT D213L CTGK279V GTG C346G GGG L015Q CAG Q081H CAT T147D GAT D213M ATG K279W TGGC346I ATT L015R CGG Q081L CTG T147F TTT D213N AAT K279Y TAT C346K AAGL015S TCG Q081M ATG T147G GGT D213P CCT F280D GAT C346L CTT L015T ACTQ081N AAT T147I ATT D213Q CAG F280E GAG C346M ATG L015V GTT Q081P CCGT147L CTT D213R CGT F280G GGG C346P CCT L015W TGG Q081R AGG T147M ATGD213S TCG F280H CAT C346Q CAG L015Y TAT Q081S TCT T147P CCT D213V GTGF280I ATT C346R CGG W016A GCG Q081V GTT T147Q CAG D213W TGG F280L TTGC346S TCT W016C TGT Q081W TGG T147R CGT D213Y TAT F280M ATG C346T ACTW016D GAT Q081Y TAT T147S AGT L214A GCG F280N AAT C346V GTG W016E GAGK082A GCT T147V GTT L214C TGT F280P CCT C346W TGG W016F TTT K082E GAGT147W TGG L214D GAT F280Q CAG Q347A GCT W016G GGT K082G GGT T147Y TATL214E GAG F280R CGT Q347C TGT W016H CAT K082H CAT E148C TGT L214G GGGF280S TCG Q347E GAG W016K AAG K082I ATT E148F TTT L214H CAT F280T ACTQ347F TTT W016L CTT K082L CTT E148G GGG L214K AAG F280V GTG Q347G GGTW016M ATG K082M ATG E148H CAT L214N AAT F280W TGG Q347I ATT W016P CCTK082N AAT E148I ATT L214P CCG L281A GCG Q347L TTG W016R CGT K082P CCTE148K AAG L214Q CAG L281D GAT Q347M ATG W016S TCG K082Q CAG E148L CTGL214R CGG L281F TTT Q347P CCT W016T ACT K082R CGT E148P CCT L214S TCGL281G GGT Q347R AGG W016Y TAT K082S AGT E148Q CAG L214T ACG L281H CATQ347S TCT A017D GAT K082T ACT E148R CGG L214V GTG L281I ATT Q347T ACTA017E GAG K082V GTG E148S TCT L214Y TAT L281K AAG Q347V GTG A017G GGGK082W TGG E148T ACT S215A GCT L281N AAT Q347W TGG A017H CAT K082Y TATE148V GTG S215C TGT L281P CCG Q347Y TAT A017I ATT I083E GAG E148W TGGS215D GAT L281Q CAG E348C TGT A017L CTT I083F TTT E148Y TAT S215E GAGL281R CGG E348D GAT A017N AAT I083G GGT A149C TGT S215G GGG L281S AGTE348G GGT A017P CCG I083H CAT A149E GAG S215H CAT L281V GTT E348H CATA017Q CAG I083K AAG A149F TTT S215K AAG L281W TGG E348I ATT A017R AGGI083L CTG A149G GGT S215L TTG L281Y TAT E348L TTG A017S TCG I083N AATA149K AAG S215M ATG S282A GCG E348M ATG A017T ACG I083P CCT A149L TTGS215P CCG S282C TGT E348P CCT A017V GTG I083Q CAA A149M ATG S215Q CAGS282D GAT E348Q CAG A017W TGG I083R CGT A149P CCT S215R CGG S282E GAGE348R CGG A017Y TAT I083S TCG A149Q CAG S215T ACT S282F TTT E348S TCTW018C TGT I083T ACT A149R CGG S215V GTG S282G GGT E348T ACT W018D GATI083V GTT A149S TCT S215W TGG S282L CTT E348V GTT W018F TTT I083Y TATA149T ACT W216D GAT S282M ATG E348W TGG W018G GGG S084D GAT A149V GTTW216E GAG S282P CCT E348Y TAT W018H CAT S084E GAG A149W TGG W216G GGTS282Q CAG Q349A GCT W018I ATT S084F TTT A149Y TAT W216H CAT S282R CGTQ349D GAT W018L CTG S084G GGT T150A GCT W216I ATT S282T ACT Q349E GAGW018M ATG S084H CAT T150C TGT W216K AAG S282V GTT Q349F TTT W018P CCGS084I ATT T150D GAT W216L CTG S282W TGG Q349G GGT W018Q CAG S084L CTTT150E GAG W216M ATG S282Y TAT Q349H CAT W018R CGG S084M ATG T150F TTTW216N AAT Q283A GCG Q349K AAG W018S AGT S084N AAT T150G GGG W216P CCTQ283C TGT Q349L CTG W018T ACG S084P CCT T150I ATT W216Q CAG Q283D GATQ349M ATG W018V GTG S084Q CAG T150L TTG W216R CGG Q283E GAG Q349N AATW018Y TAT S084R CGG T150N AAT W216T ACG Q283F TTT Q349P CCT N019A GCGS084T ACT T150P CCT W216V GTG Q283G GGG Q349R CGT N019C TGT S084W TGGT150R AGG W216Y TAT Q283H CAT Q349S TCG N019F TTT S084Y TAT T150S TCTL217A GCG Q283L CTT Q349T ACT N019G GGG L085A GCT T150V GTG L217C TGTQ283N AAT Q349V GTG N019H CAT L085C TGT T150W TGG L217E GAG Q283P CCGQ349W TGG N019I ATT L085D GAT T150Y TAT L217G GGT Q283R CGT Q349Y TATN019L CTG L085E GAG E151A GCT L217H CAT Q283S TCT G350A GCT N019M ATGL085F TTT E151C TGT L217I ATT Q283T ACT G350D GAT N019P CCG L085G GGGE151G GGT L217M ATG Q283W TGG G350E GAG N019Q CAG L085H CAT E151H CATL217P CCG Q283Y TAT G350F TTT N019R CGT L085K AAG E151K AAG L217Q CAGD284A GCT G350H CAT N019S TCG L085N AAT E151L TTG L217R AGG D284C TGTG350K AAG N019V GTT L085P CCT E151M ATG L217S TCT D284E GAG G350L CTGN019W TGG L085Q CAG E151N AAT L217T ACG D284G GGT G350M ATG N019Y TATL085R CGT E151Q CAG L217V GTG D284H CAT G350N AAT A020D GAT L085S TCGE151R AGG L217W TGG D284I ATT G350P CCT A020E GAG L085T ACT E151S TCGL217Y TAT D284L TTG G350R CGT A020F TTT L085V GTT E151T ACT W218A GCTD284M ATG G350S TCT A020G GGG Q086A GCT E151V GTT W218D GAT D284N AATG350T ACT A020H CAT Q086C TGT E151W TGG W218F TTT D284P CCG G350V GTGA020K AAG Q086D GAT E151Y TAT W218G GGT D284Q CAG G350Y TAT A020L CTGQ086E GAG K152A GCT W218H CAT D284S TCT V351A GCT A020N AAT Q086F TTTK152C TGT W218I ATT D284T ACG V351C TGT A020P CCG Q086G GGT K152F TTTW218K AAG D284V GTT V351D GAT A020Q CAG Q086H CAT K152G GGT W218L CTTD284Y TAT V351E GAG A020R CGT Q086I ATT K152I ATT W218M ATG E285A GCGV351F TTT A020S TCT Q086K AAG K152L TTG W218P CCT E285F TTT V351G GGTA020T ACT Q086L CTG K152M ATG W218Q CAG E285G GGG V351H CAT A020V GTTQ086M ATG K152N AAT W218R CGG E285H CAT V351I ATT A020Y TAT Q086N AATK152P CCT W218S TCG E285K AAG V351L TTG P021A GCG Q086P CCT K152R AGGW218T ACT E285M ATG V351N AAT P021C TGT Q086R CGG K152S TCT W218V GTGE285N AAT V351Q CAG P021D GAT Q086S TCT K152T ACT N219A GCG E285P CCTV351R AGG P021E GAG Q086T ACT K152V GTG N219C TGT E285Q CAG V351S TCTP021G GGG Q086V GTG K152W TGG N219D GAT E285R CGT V351W TGG P021H CATQ086W TGG K152Y TAT N219E GAG E285S AGT V351Y TAT P021I ATT D087A GCTA153C TGT N219G GGG E285T ACG C352A GCT P021K AAG D087C TGT A153E GAGN219H CAT E285V GTG C352D GAT P021L CTT D087E GAG A153F TTT N219I ATTE285W TGG C352E GAG P021M ATG D087G GGG A153G GGT N219K AAG E285Y TATC352F TTT P021R CGT D087H CAT A153H CAT N219L CTT L286A GCG C352G GGGP021S TCT D087I ATT A153I ATT N219M ATG L286C TGT C352K AAG P021T ACGD087L CTG A153K AAG N219P CCT L286D GAT C352M ATG P021V GTT D087M ATGA153L CTG N219R CGT L286E GAG C352P CCT P021W TGG D087P CCT A153M ATGN219S TCG L286F TTT C352Q CAG S022A GCT D087Q CAG A153P CCT N219T ACTL286G GGT C352R CGT S022C TGT D087R AGG A153Q CAG N219W TGG L286H CATC352S AGT S022D GAT D087S TCG A153R CGT E220A GCG L286K AAG C352T ACTS022E GAG D087T ACT A153S AGT E220D GAT L286M ATG C352V GTG S022G GGGD087V GTT A153T ACT E220G GGG L286P CCT C352W TGG S022H CAT D087Y TATA153V GTG E220H CAT L286R AGG C352Y TAT S022K AAG H088A GCT A153W TGGE220I ATT L286S AGT I353A GCT S022L CTG H088C TGT K154A GCT E220K AAGL286T ACG I353C TGT S022M ATG H088E GAG K154C TGT E220L TTG L286W TGGI353E GAG S022N AAT H088F TTT K154D GAT E220M ATG L286Y TAT I353F TTTS022P CCG H088G GGG K154E GAG E220N AAT V287A GCT I353G GGG S022R CGGH088I ATT K154G GGT E220P CCG V287C TGT I353H CAT S022T ACT H088K AAGK154H CAT E220R CGG V287D GAT I353K AAG S022V GTG H088L TTG K154I ATTE220S TCT V287E GAG I353L CTT S022Y TAT H088M ATG K154L CTG E220T ACGV287F TTT I353M ATG E023A GCT H088P CCT K154P CCT E220V GTG V287G GGGI353Q CAG E023D GAT H088R CGT K154R CGG E220W TGG V287I ATT I353R CGTE023F TTT H088S AGT K154S AGT S221A GCG V287K AAG I353S TCG E023G GGGH088T ACT K154T ACT S221C TGT V287L CTT I353T ACT E023H CAT H088V GTTK154V GTG S221D GAT V287N AAT I353V GTG E023L CTT H088Y TAT K154W TGGS221E GAG V287P CCT I353W TGG E023M ATG L089A GCT K154Y TAT S221G GGGV287Q CAG R354C TGT E023N AAT L089C TGT Q155A GCT S221H CAT V287R CGGR354D GAT E023P CCT L089D GAT Q155C TGT S221I ATT V287S TCT R354E GAGE023Q CAG L089E GAG Q155D GAT S221K AAG V287T ACT R354G GGT E023R CGGL089G GGG Q155F TTT S221L TTG Y288D GAC R354H CAT E023S TCT L089K AAGQ155G GGG S221M ATG Y288E GAG R354I ATT E023T ACG L089M ATG Q155H CATS221P CCG Y288F TTT R354K AAG E023V GTG L089N AAT Q155K AAG S221Q CAGY288G GGG R354L CTT E023W TGG L089P CCT Q155L CTT S221R CGG Y288H CATR354M ATG F024A GCG L089Q CAG Q155M ATG S221T ACT Y288I ATT R354P CCTF024C TGT L089R AGG Q155P CCT S221V GTG Y288K AAG R354Q CAG F024E GAGL089S TCG Q155R CGG T222A GCG Y288L CTG R354S TCT F024G GGG L089T ACTQ155S AGT T222D GAT Y288P CCT R354V GTG F024H CAT L089W TGG Q155T ACTT222E GAG Y288Q CAG R354W TGG F024I ATT L089Y TAT Q155V GTT T222F TTTY288R CGT R354Y TAT F024K AAG D090A GCT Q155W TGG T222G GGG Y288S TCTK355D GAT F024L TTG D090C TGT Q155Y TAT T222I ATT Y288T ACT K355F TTTF024M ATG D090E GAG E156A GCT T222K AAA Y288V GTG K355G GGG F024N AATD090G GGG E156C TGT T222L TTG Y288W TGG K355H CAT F024P CCT D090H CATE156D GAT T222N AAT T289A GCT K355L CTG F024R CGT D090I ATT E156G GGTT222P CCG T289C TGT K355M ATG F024T ACG D090K AAG E156I ATT T222R CGGT289E GAG K355N AAT F024V GTT D090L CTT E156K AAG T222S AGT T289G GGTK355P CCT F024Y TAT D090N AAT E156L CTG T222V GTT T289H CAT K355Q CAGC025D GAT D090P CCT E156M ATG T222W TGG T289K AAG K355R CGT C025E GAGD090Q CAG E156P CCT T222Y TAT T289L CTT K355S TCT C025F TTT D090R AGGE156Q CAG A223C TGT T289M ATG K355T ACT C025G GGG D090S AGT E156R CGGA223D GAT T289N AAT K355V GTG C025H CAT D090T ACT E156S TCT A223E GAGT289P CCT K355W TGG C025I ATT D090W TGG E156T ACT A223G GGG T289Q CAGK355Y TAT C025K AAG K091A GCT E156V GTT A223H CAT T289R AGG N356A GCTC025L TTG K091D GAT E156W TGG A223K AAG T289S TCG N356C TGT C025N AATK091E GAG F157A GCT A223L CTG T289V GTG N356D GAT C025P CCT K091F TTTF157C TGT A223P CCT T289Y TAT N356F TTT C025R CGT K091G GGG F157D GATA223Q CAG F290A GCT N356G GGG C025S TCT K091H CAT F157E GAG A223R AGGF290C TGT N356H CAT C025T ACT K091I ATT F157G GGT A223S TCT F290D GATN356K AAG C025V GTG K091L TTG F157H CAT A223T ACG F290G GGG N356L CTGC025Y TAT K091N AAT F157I ATT A223V GTG F290H CAT N356P CCT L026A GCTK091Q CAG F157K AAG A223W TGG F290I ATT N356Q CAG L026E GAG K091R CGTF157L TTG A223Y TAT F290K AAG N356R CGG L026G GGT K091S TCT F157M ATGL224A GCT F290L TTG N356S AGT L026H CAT K091T ACT F157P CCT L224D GATF290M ATG N356T ACT L026I ATT K091Y TAT F157Q CAG L224E GAG F290Q CAGN356V GTG L026K AAG A092C TGT F157R CGG L224F TTT F290R AGG N356W TGGL026M ATG A092E GAG F157S TCG L224G GGG F290S TCG W357A GCT L026P CCGA092F TTT F157T ACT L224I ATT F290T ACT W357C TGT L026Q CAG A092G GGTF157V GTG L224M ATG F290V GTT W357D GAT L026R CGG A092H CAT F157W TGGL224P CCG F290Y TAT W357E GAG L026S TCT A092K AAG E158A GCT L224Q CAGG291A GCT W357F TTT L026T ACT A092L CTG E158C TGT L224R AGG G291C TGTW357G GGG L026V GTT A092M ATG E158D GAT L224S AGT G291D GAT W357K AAGL026W TGG A092P CCT E158F TTT L224T ACT G291E GAG W357L TTG L026Y TATA092Q CAG E158G GGG L224V GTT G291F TTT W357M ATG G027A GCT A092R CGTE158H CAT L224W TGG G291H CAT W357P CCT G027C TGT A092T ACT E158K AAGL224Y TAT G291L CTG W357Q CAG G027D GAT A092V GTT E158L CTG Y225A GCGG291M ATG W357R CGT G027E GAG A092W TGG E158N AAT Y225D GAT G291N AATW357S AGT G027F TTT A092Y TAT E158P CCT Y225E GAG G291P CCT W357T ACTG027H CAT K093D GAT E158Q CAG Y225G GGT G291Q CAG W357V GTG G027I ATTK093E GAG E158R CGG Y225H CAT G291R CGG N358C TGT G027K AAG K093F TTTE158S TCG Y225K AAG G291S TCT N358D GAT G027L CTG K093G GGT E158V GTGY225L CTG G291T ACT N358E GAG G027P CCT K093H CAT E158Y TAT Y225P CCGG291V GTG N358G GGG G027Q CAG K093I ATT K159A GCT Y225Q CAG G291W TGGN358H CAT G027R CGG K093L CTG K159D GAT Y225R AGG G291Y TAT N358I ATTG027S TCG K093M ATG K159E GAG Y225S TCT E292A GCT N358K AAG G027T ACTK093N AAT K159F TTT Y225T ACG E292C TGT N358L CTG G027W TGG K093P CCTK159G GGT Y225V GTG E292F TTT N358P CCT K028A GCG K093Q CAG K159H CATY225W TGG E292G GGT N358Q CAG K028D GAT K093R CGG K159L CTT P226A GCGE292H CAT N358R CGT K028E GAG K093S AGT K159M ATG P226C TGT E292I ATTN358S TCT K028F TTT K093T ACT K159N AAT P226D GAT E292K AAG N358T ACTK028G GGG K093V GTT K159Q CAG P226E GAG E292L TTG N358V GTG K028I ATTK094A GCT K159R CGG P226F TTT E292N AAT N358W TGG K028L TTG K094C TGTK159S TCT P226G GGT E292P CCT S359A GCT K028M ATG K094D GAT K159V GTGP226L CTT E292Q CAG S359C TGT K028N AAT K094E GAG K159W TGG P226N AATE292R CGG S359D GAT K028P CCT K094F TTT K159Y TAT P226Q CAG E292T ACTS359E GAG K028R CGG K094G GGG A160C TGT P226R AGG E292V GTT S359F TTTK028S AGT K094H CAT A160F TTT P226S TCT E292W TGG S359G GGG K028T ACTK094L TTG A160G GGG P226T ACG T293A GCT S359H CAT K028V GTT K094M ATGA160H CAT P226V GTT T293C TGT S359K AAG K028W TGG K094N AAT A160I ATTP226W TGG T293D GAT S359L TTG F029A GCT K094P CCT A160K AAG P226Y TATT293E GAG S359M ATG F029C TGT K094Q CAG A160L CTG S227A GCT T293F TTTS359P CCT F029E GAG K094R AGG A160M ATG S227F TTT T293G GGT S359R CGGF029G GGG K094S TCT A160N AAT S227G GGG T293K AAG S359T ACT F029H CATK094T ACT A160Q CAG S227H CAT T293L CTT S359V GTT F029I ATT D095A GCTA160R AGG S227I ATT T293M ATG S359W TGG F029K AAG D095C TGT A160S AGTS227K AAG T293N AAT S360A GCT F029L CTT D095E GAG A160V GTG S227L TTGT293P CCT S360C TGT F029M ATG D095F TTT A160W TGG S227M ATG T293Q CAGS360E GAG F029P CCG D095G GGG A160Y TAT S227P CCT T293S TCT S360F TTTF029R CGG D095H CAT G161A GCT S227Q CAG T293V GTG S360G GGG F029S TCGD095K AAG G161C TGT S227R CGG T293Y TAT S360I ATT F029T ACG D095L TTGG161D GAT S227T ACG V294A GCT S360K AAG F029V GTG D095M ATG G161E GAGS227V GTG V294C TGT S360L CTG F029W TGG D095P CCT G161H CAT S227W TGGV294E GAG S360M ATG D030A GCG D095Q CAG G161I ATT S227Y TAT V294G GGGS360N AAT D030E GAG D095S TCT G161K AAG I228A GCG V294H CAT S360P CCTD030F TTT D095V GTG G161L CTT I228E GAG V294K AAG S360Q CAG D030G GGGD095W TGG G161M ATG I228F TTT V294L TTG S360R AGG D030H CAT D095Y TATG161Q CAG I228G GGG V294M ATG S360T ACT D030K AAG I096A GCT G161R CGTI228H CAT V294N AAT S360V GTT D030L TTG I096C TGT G161S AGT I228K AAGV294P CCT D361A GCT D030M ATG I096D GAT G161T ACT I228L TTG V294Q CAGD361C TGT D030P CCT I096E GAG G161V GTG I228M ATG V294R AGG D361E GAGD030Q CAG I096F TTT G161W TGG I228N AAT V294S AGT D361G GGG D030R CGGI096G GGG K162A GCT I228P CCG V294T ACT D361H CAT D030S TCG I096H CATK162D GAT I228Q CAG V294W TGG D361L TTG D030T ACT I096L TTG K162E GAGI228R CGT A295C TGT D361M ATG D030V GTT I096N AAT K162F TTT I228S TCTA295D GAT D361N AAT D030W TGG I096P CCT K162G GGG I228T ACT A295E GAGD361P CCT E031A GCG I096R CGT K162H CAT I228W TGG A295F TTT D361Q CAGE031C TGT I096S AGT K162L TTG Y229E GAG A295G GGG D361R AGG E031G GGGI096T ACT K162M ATG Y229F TTT A295H CAT D361S TCG E031H CAT I096V GTGK162P CCT Y229G GGT A295I ATT D361V GTT E031I ATT I096W TGG K162Q CAGY229H CAT A295L CTG D361W TGG E031K AAG T097A GCT K162R CGG Y229I ATTA295N AAT D361Y TAT E031L CTG T097C TGT K162S TCG Y229K AAG A295P CCTY362A GCT E031N AAC T097D GAT K162V GTG Y229L TTG A295Q CAG Y362C TGTE031P CCG T097E GAG K162W TGG Y229N AAT A295S AGT Y362E GAG E031R CGGT097F TTT K162Y TAT Y229P CCT A295T ACT Y362G GGG E031S TCT T097G GGGD163A GCT Y229Q CAG A295V GTT Y362H CAT E031T ACG T097I ATT D163C TGTY229R CGT A295Y TAT Y362K AAG E031V GTG T097L CTT D163E GAG Y229S TCGL296A GCT Y362L CTT E031W TGG T097N AAT D163F TTT Y229T ACT L296C TGTY362M ATG E031Y TAT T097P CCT D163G GGG Y229V GTG L296F TTT Y362N AATP032A GCG T097Q CAG D163H CAC Y229W TGG L296G GGT Y362P CCT P032C TGTT097R CGG D163K AAG L230A GCG L296I ATT Y362R CGG P032F TTT T097S TCGD163L CTT L230E GAG L296K AAG Y362S AGT P032G GGG T097W TGG D163P CCTL230G GGG L296M ATG Y362T ACT P032H CAT T097Y TAT D163Q CAG L230H CATL296P CCT Y362V GTG P032K AAG F098A GCT D163R AGG L230I ATT L296Q CAGY362W TGG P032L CTG F098C TGT D163S TCG L230K AAG L296R CGT L363A GCTP032M ATG F098D GAT D163T ACT L230M ATG L296S TCG L363C TGT P032N AATF098E GAG D163V GTG L230N AAT L296T ACT L363D GAT P032Q CAG F098G GGGD163W TGG L230P CCT L296V GTT L363E GAG P032R CGG F098H CAT F164A GCTL230R CGT L296W TGG L363F TTT P032S TCG F098I ATT F164C TGT L230S AGTL296Y TAT L363G GGG P032T ACT F098L TTG F164D GAT L230T ACT G297A GCTL363H CAT P032V GTG F098M ATG F164E GAG L230V GTT G297C TGT L363I ATTP032W TGG F098P CCT F164G GGG L230W TGG G297E GAG L363P CCT P032Y TATF098Q CAG F164H CAT L230Y TAT G297H CAT L363Q CAG L033C TGT F098R CGTF164L TTG N231A GCT G297I ATT L363R CGG L033D GAT F098S TCG F164M ATGN231C TGT G297L CTT L363S TCG L033G GGG F098V GTT F164N AAT N231D GATG297N AAT L363T ACT L033H CAT F098W TGG F164P CCT N231F TTT G297P CCTL363V GTG L033I ATT Y099A GCT F164Q CAG N231G GGG G297Q CAG L363W TGGL033M ATG Y099C TGT F164R CGG N231H CAT G297R CGG H364A GCT L033N AATY099E GAG F164S AGT N231I ATT G297S AGT H364C TGT L033P CCG Y099F TTTF164V GTT N231K AAG G297T ACT H364D GAT L033Q CAG Y099G GGT F164W TGGN231L CTT G297V GTG H364E GAG L033R AGG Y099I ATT L165A GCT N231P CCTG297W TGG H364F TTT L033S TCG Y099L TTG L165C TGT N231Q CAG G297Y TATH364G GGG L033T ACT Y099N AAT L165D GAT N231R CGT A298C TGT H364K AAGL033V GTT Y099P CCT L165F TTT N231S TCT A298E GAG H364L CTG L033W TGGY099Q CAG L165G GGG N231T ACG A298G GGG H364M ATG L033Y TAT Y099R AGGL165H CAT N231V GTG A298I ATT H364P CCT D034A GCT Y099S TCG L165N AATT232A GCG A298L TTG H364R CGG D034E GAG Y099T ACT L165P CCT T232C TGTA298M ATG H364S TCT D034G GGT Y099V GTT L165Q CAG T232F TTT A298N AATH364T ACT D034H CAT Y099W TGG L165R CGG T232G GGG A298P CCT H364V GTGD034I ATT M100C TGT L165S TCG T232H CAT A298Q CAG H364Y TAT D034K AAGM100E GAG L165T ACT T232K AAG A298R CGT L365A GCT D034L CTT M100F TTTL165V GTG T232L CTT A298S TCG L365C TGT D034N AAT M100G GGT L165W TGGT232M ATG A298T ACT L365D GAT D034P CCT M100K AAG L165Y TAT T232N AATA298V GTG L365E GAG D034Q CAG M100L CTG V166A GCT T232P CCG A298W TGGL365G GGG D034R CGT M100N AAT V166C TGT T232Q CAG A298Y TAT L365I ATTD034S AGT M100P CCT V166D GAT T232R AGG S299A GCT L365M ATG D034T ACGM100Q CAG V166E GAG T232S AGT S299C TGT L365N AAT D034V GTT M100R CGGV166F TTT T232V GTG S299D GAT L365P CCT D034W TGG M100S TCT V166G GGTT232Y TAT S299E GAG L365Q CAG M035A GCG M100T ACT V166H CAT Q233A GCGS299F TTT L365R CGG M035D GAT M100V GTT V166L CTT Q233C TGT S299G GGGL365S AGT M035F TTT M100W TGG V166N AAT Q233D GAT S299H CAT L365T ACTM035G GGG M100Y TAT V166P CCT Q233F TTT S299I ATT L365V GTG M035H CATP101A GCT V166Q CAG Q233G GGG S299L CTT L365W TGG M035I ATT P101C TGTV166R CGG Q233I ATT S299M ATG L365Y TAT M035L TTG P101F TTT V166T ACTQ233K AAG S299P CCT N366A GCT M035N AAT P101G GGG V166W TGG Q233L CTGS299Q CAG N366C TGT M035P CCG P101H CAT V166Y TAT Q233P CCG S299R AGGN366E GAG M035Q CAG P101I ATT E167A GCT Q233R AGG S299T ACT N366F TTTM035R CGT P101K AAG E167D GAT Q233S TCG S299Y TAT N366G GGG M035S TCTP101L CTT E167F TTT Q233T ACG G300A GCT N366K AAG M035T ACT P101M ATGE167G GGT Q233V GTG G300C TGT N366L TTG M035V GTT P101N AAT E167H CATQ233W TGG G300D GAT N366M ATG M035Y TAT P101Q CAG E167K AAG Q233Y TATG300E GAG N366P CCT S036A GCG P101R AGG E167L TTG Q234A GCT G300F TTTN366Q CAG S036C TGT P101S TCT E167M ATG Q234C TGT G300L CTT N366R AGGS036D GAT P101T ACT E167N AAT Q234D GAT G300M ATG N366S TCT S036F TTTP101Y TAT E167P CCT Q234E GAG G300N AAT N366T ACT S036G GGT V102A GCTE167R AGG Q234G GGT G300P CCT N366V GTT S036H CAT V102C TGT E167S TCGQ234H CAT G300Q CAG N366W TGG S036K AAG V102E GAG E167T ACT Q234L CTTG300R AGG P367A GCT S036L TTG V102G GGT E167V GTT Q234M ATG G300S TCGP367C TGT S036N AAT V102H CAT E167Y TAT Q234N AAT G300T ACT P367E GAGS036P CCG V102K AAG T168A GCT Q234P CCG G300V GTT P367F TTT S036R CGGV102L TTG T168C TGT Q234R CGG G300W TGG P367G GGT S036T ACG V102M ATGT168D GAT Q234S AGT I301A GCT P367H CAT S036V GTT V102N AAT T168E GAGQ234T ACT I301E GAG P367I ATT S036W TGG V102P CCT T168F TTT Q234V GTGI301G GGG P367K AAG S036Y TAT V102Q CAG T168G GGG Q234W TGG I301H CATP367L CTG L037A GCG V102R AGG T168H CAT S235A GCG I301K AAG P367M ATGL037C TGT V102S TCT T168K AAG S235E GAG I301L CTG P367Q CAG L037E GAGV102T ACT T168L CTG S235F TTT I301M ATG P367R CGT L037F TTT V102W TGGT168P CCT S235G GGG I301N AAT P367S TCG L037G GGG D103A GCT T168R CGGS235H CAT I301P CCT P367V GTT L037I ATT D103E GAG T168S TCT S235K AAGI301Q CAG P367W TGG L037K AAG D103F TTT T168V GTG S235L CTT I301R CGGD368A GCT L037M ATG D103G GGG T168W TGG S235M ATG I301S AGT D368C TGTL037N AAT D103H CAT T168Y TAT S235P CCT I301V GTT D368E GAG L037P CCTD103I ATT I169A GCT S235Q CAG I301W TGG D368G GGT L037R AGG D103L CTTI169D GAT S235R CGG I301Y TAT D368H CAT L037S TCT D103N AAT I169F TTTS235T ACG V302C TGT D368K AAG L037T ACG D103Q CAG I169G GGG S235V GTGV302D GAT D368L CTT L037V GTG D103R AGG I169H CAT S235W TGG V302E GAGD368M ATG L037W TGG D103S TCG I169K AAG S235Y TAT V302F TTT D368P CCTF038A GCG D103T ACT I169L TTG P236A GCT V302G GGT D368R CGT F038C TGTD103V GTT I169N AAT P236C TGT V302H CAT D368S AGT F038E GAG D103W TGGI169P CCT P236E GAG V302I ATT D368T ACT F038G GGG D103Y TAT I169Q CAGP236G GGG V302L TTG D368V GTT F038K AAG N104A GCT I169R CGG P236H CATV302M ATG D368W TGG F038L CTT N104C TGT I169S TCG P236I ATT V302P CCTD368Y TAT F038M ATG N104F TTT I169T ACT P236K AAG V302R AGG N369A GCTF038N AAT N104G GGG I169V GTT P236L CTG V302S TCG N369C TGT F038P CCTN104H CAT I169Y TAT P236N AAT V302T ACT N369E GAG F038Q CAG N104I ATTK170A GCT P236Q CAG V302W TGG N369F TTT F038R AGG N104K AAG K170C TGTP236R CGT V302Y TAT N369H CAT F038S TCT N104L CTG K170D GAT P236S AGTI303A GCT N369I ATT F038T ACT N104M ATG K170E GAG P236T ACT I303C TGTN369K AAG F038W TGG N104P CCT K170G GGG P236W TGG I303D GAT N369L CTTF038Y TAT N104R AGG K170I ATT P236Y TAT I303E GAG N369P CCT S039A GCGN104S TCT K170L TTG V237A GCG I303F TTT N369Q CAG S039C TGT N104T ACTK170M ATG V237C TGT I303G GGT N369R CGG S039D GAT N104V GTT K170N AATV237E GAG I303K AAG N369S TCG S039F TTT N104W TGG K170P CCT V237F TTTI303L TTG N369T ACT S039G GGT L105A GCT K170Q CAG V237G GGT I303M ATGN369V GTG S039L TTG L105C TGT K170R CGT V237H CAT I303P CCT N369W TGGS039M ATG L105D GAT K170V GTT V237L TTG I303R CGT F370A GCT S039N AATL105E GAG K170W TGG V237N AAT I303S AGT F370D GAT S039P CCG L105G GGTK170Y TAT V237P CCT I303V GTG F370E GAG S039Q CAG L105H CAT L171A GCTV237Q CAG I303W TGG F370G GGG S039R CGT L105I ATT L171C TGT V237R CGGI303Y TAT F370H CAT S039T ACT L105M ATG L171D GAT V237S TCG W304A GCTF370I ATT S039V GTT L105N AAT L171G GGG V237T ACG W304C TGT F370K AAGS039W TGG L105P CCT L171H CAT V237W TGG W304D GAT F370L CTG S039Y TATL105Q CAG L171I ATT V237Y TAT W304G GGT F370N AAT F040A GCG L105R CGGL171M ATG A238D GAT W304I ATT F370P CCT F040D GAT L105S TCT L171N AATA238E GAG W304L CTG F370Q CAG F040E GAG L105T ACT L171P CCT A238F TTTW304M ATG F370R AGG F040G GGT L105V GTT L171Q CAG A238G GGT W304N AATF370S TCT F040I ATT L105W TGG L171R CGT A238H CAT W304P CCT F370V GTGF040K AAG G106A GCT L171S AGT A238K AAG W304Q CAG F370Y TAT F040L CTGG106C TGT L171V GTG A238L CTT W304R CGG A371C TGT F040N AAT G106D GATL171W TGG A238P CCG W304S AGT A371E GAG F040Q CAG G106E GAG L171Y TATA238Q CAG W304T ACT A371F TTT F040R CGG G106F TTT G172A GCT A238R AGGW304V GTG A371G GGG F040S TCT G106H CAT G172C TGT A238S AGT W304Y TATA371H CAT F040T ACT G106I ATT G172D GAT A238T ACG G305C TGT A371I ATTF040V GTT G106L CTG G172E GAG A238V GTG G305D GAT A371K AAG F040W TGGG106M ATG G172I ATT A238W TGG G305E GAG A371L CTT F040Y TAT G106N AATG172L CTT A238Y TAT G305F TTT A371M ATG I041A GCG G106P CCT G172M ATGA239C TGT G305H CAT A371P CCT I041C TGT G106S AGT G172P CCT A239F TTTG305K AAG A371R CGT I041D GAT G106V GTG G172Q CAG A239G GGT G305L CTTA371S TCG I041E GAG G106W TGG G172R CGT A239H CAT G305N AAT A371T ACTI041F TTT G106Y TAT G172S TCT A239I ATT G305P CCT A371V GTG I041G GGGM107A GCT G172T ACT A239K AAG G305Q CAG A371W TGG I041H CAT M107C TGTG172V GTT T240K AAG G305R CGT I372A GCT I041N AAT M107D GAT G172W TGGA239L TTG G305S TCG I372D GAT I041P CCG M107F TTT G172Y TAT A239N AATG305T ACT I372E GAG I041Q CAG M107G GGG K173D GAT A239P CCT G305V GTGI372F TTT I041R AGG M107H CAT K173E GAG A239R AGG G305Y TAT I372G GGTI041S TCT M107I ATT K173G GGG A239S TCT T306A GCT I372H CAT I041T ACGM107K AAG K173H CAT A239T ACT T306C TGT I372K AAG I041V GTT M107L CTTK173I ATT A239V GTT T306D GAT I372L CTG I041W TGG M107P CCT K173L CTTA239W TGG T306E GAG I372N AAT G042A GCT M107Q CAG K173M ATG A239Y TATT306F TTT I372P CCT G042C TGT M107R CGT K173N AAT T240A GCG T306G GGTI372R CGG G042D GAT M107S TCT K173P CCT T240E GAG T306H CAT I372S TCTG042E GAG M107V GTT K173Q CAG T240F TTT T306I ATT I372T ACT G042H CATM107W TGG K173R CGG T240G GGG T306L CTG I372V GTG G042I ATT A108D GATK173S TCG T240L CTT T306P CCT I372W TGG G042K AAG A108E GAG K173V GTGT240M ATG T306R AGG Q373A GCT G042L CTG A108F TTT K173W TGG T240N AATT306S AGT Q373C TGT G042M ATG A108G GGT K173Y TAT T240P CCT T306V GTGQ373E GAG G042P CCT A108H CAT L174A GCT T240Q CAG T306W TGG Q373F TTTG042Q CAG A108K AAG L174C TGT T240R CGT T306Y TAT Q373G GGT G042R CGGA108L TTG L174G GGG T240S AGT L307C TGT Q373H CAT G042S TCT A108M ATGL174H CAT T240V GTG L307E GAG Q373K AAG G042T ACT A108N AAT L174K AAGT240W TGG L307F TTT Q373L CTG G042V GTT A108P CCT L174M ATG T240Y TATL307G GGG Q373M ATG S043A GCG A108Q CAG L174N AAT L241A GCG L307I ATTQ373N AAT S043D GAT A108R CGG L174P CCT L241C TGT L307K AAG Q373P CCTS043E GAG A108S TCT L174Q CAG L241D GAT L307N AAT Q373R CGT S043F TTTA108T ACT L174R CGT L241E GAG L307P CCT Q373S TCT S043G GGT A108V GTGL174S TCG L241F TTT L307Q CAG Q373T ACT S043H CAT A108Y TAT L174T ACTL241G GGG L307R AGG Q373V GTT S043I ATT V109A GCT L174V GTT L241I ATTL307S AGT Q373W TGG S043K AAG V109C TGT L174W TGG L241K AAG L307T ACTL374A GCT S043L CTT V109D GAT L174Y TAT L241P CCT L307V GTG L374D GATS043N AAT V109E GAG L175C TGT L241Q CAG L307W TGG L374E GAG S043P CCTV109F TTT L175D GAT L241R CGG L307Y TAT L374G GGT S043Q CAG V109G GGGL175E GAG L241S TCT S308C TGT L374H CAT S043R CGG V109H CAT L175F TTTL241T ACG S308D GAT L374I ATT S043T ACT V109L TTG L175G GGG L241V GTTS308F TTT L374M ATG S043V GTG V109M ATG L175H CAT L241W TGG S308G GGTL374N AAT P044A GCT V109P CCT L175K AAG Y242A GCG S308H CAT L374P CCTP044C TGT V109Q CAG L175N AAT Y242C TGT S308K AAG L374R AGG P044E GAGV109R AGG L175P CCT Y242D GAT S308L CTG L374S AGT P044F TTT V109T ACTL175R CGT Y242F TTT S308M ATG L374T ACT P044G GGG V109W TGG L175S TCTY242G GGT S308N AAT L374V GTG P044H CAT V109Y TAT L175T ACT Y242I ATTS308P CCT L374W TGG P044I ATT I110A GCT L175V GTG Y242K AAG S308R CGGL374Y TAT P044L CTT I110C TGT L175W TGG Y242L CTT S308T ACT E375A GCTP044N AAT I110D GAT L175Y TAT Y242M ATG S308V GTT E375C TGT P044Q CAGI110F TTT R176A GCT Y242P CCG S308W TGG E375F TTT P044R CGT I110G GGGR176C TGT Y242R CGG S308Y TAT E375G GGT P044S TCT I110H CAT R176E GAGY242S TCT I309D GAT E375I ATT P044T ACT I110K AAG R176F TTT Y242T ACGI309E GAG E375K AAG P044W TGG I110L CTG R176G GGG Y242V GTT I309G GGTE375L CTT P044Y ACG I110M ATG R176H CAT Y242W TGG I309H CAT E375M ATGR045A GCG I110N AAT R176I ATT V243A GCG I309K AAG E375N AAT R045D GATI110P CCT R176K AAG V243C TGT I309L CTG E375P CCT R045F TTT I110R CGTR176L CTT V243D GAT I309M ATG E375R CGT R045G GGG I110S AGT R176P CCTV243F TTT I309N AAT E375S TCT R045H CAT I110V GTT R176Q CAG V243G GGGI309Q CAG E375T ACT R045I ATT I110W TGG R176S AGT V243H CAT I309R CGTE375V GTT R045K AAG D111C TGT R176T ACT V243L CTT I309S AGT E375Y TATR045M ATG D111E GAG R176V GTG V243M ATG I309T ACT K376A GCT R045P CCTD111G GGT R176W TGG V243P CCT I309V GTG K376D GAT R045Q CAG D111H CATP177A GCT V243Q CAG I309W TGG K376E GAG R045S TCG D111I ATT P177C TGTV243R AGG I309Y TAT K376G GGG R045T ACG D111K AAG P177D GAT V243S AGTM310A GCT K376I ATT R045V GTG D111L TTG P177F TTT V243T ACG M310C TGTK376L TTG R045W TGG D111M ATG P177G GGG V243W TGG M310E GAG K376M ATGR045Y TAT D111P ACT P177H CAT V243Y TAT M310F TTT K376P CCT I046A GCGD111Q CAG P177L CTT R244A GCG M310G GGG K376Q CAG I046C TGT D111R CGGP177M ATG R244D GAT M310K AAG K376R CGT I046E GAG D111S AGT P177Q CAGR244G GGG M310L CTG K376S AGT I046F TTT D111T ACT P177R CGG R244H CATM310N AAT K376T ACT I046H CAT D111V GTT P177S TCT R244I ATT M310P CCTK376V GTG I046L CTT D111W TGG P177T ACT R244K AAG M310Q CAG K376W TGGI046M ATG D111Y TAT P177V GTT R244M ATG M310R CGG K376Y TAT I046N AATW112C TGT P177W TGG R244N AAT M310S AGT G377C TGT I046P CCT W112D GATP177Y TAT R244P CCT M310V GTG G377D GAT I046R CGT W112E GAG N178A GCTR244Q CAG M310W TGG G377E GAG I046S TCT W112F TTT N178D GAT R244S TCTM310Y TAT G377F TTT I046T ACT W112G GGG N178E GAG R244T ACG R311A GCTG377H CAT I046V GTT W112H CAT N178G GGG R244V GTG R311C TGT G377I ATTI046W TGG W112I ATT N178I ATT R244W TGG R311E GAG G377K AAG I046Y TATW112L CTT N178K AAG R244Y TAT R311F TTT G377L CTT N047A GCT W112N AATN178L TTG N245A GCG R311G GGT G377M ATG N047D GAT W112P CCT N178M ATGN245C TGT R311H CAT G377P CCT N047F TTT W112Q CAG N178P CCT N245F TTTR311I ATT G377R AGG N047G GGG W112R CGT N178R CGG N245G GGG R311K AAGG377S TCG N047H CAT W112S TCT N178S AGT N245H CAT R311L TTG G377T ACTN047I ATT W112V GTT N178T ACT N245I ATT R311P CCT G377V GTG N047K AAGW112Y TAT N178V GTG N245K AAG R311Q CAG G377Y TAT N047L CTT E113A GCTN178W TGG N245L CTG R311S TCT G378D GAT N047M ATG E113C TGT N178Y TATN245P CCG R311T ACT G378E GAG N047P CCT E113D GAT H179A GCT N245Q CAGR311V GTG G378F TTT N047Q CAG E113F TTT H179C TGT N245R CGG R311W TGGG378I ATT N047R CGG E113G GGG H179E GAG N245S TCG S312A GCT G378K AAGN047S TCT E113H CAT H179G GGG N245T ACG S312C TGT G378L CTG N047T ACGE113L CTT H179I ATT N245V GTG S312E GAG G378M ATG N047V GTG E113P CCTH179K AAG N245W TGG S312F TTT G378N AAT N047W TGG E113Q CAG H179L CTGR246A GCG S312G GGG G378Q CAG N047Y TAT E113R CGT H179M ATG R246C TGTS312H CAT G378R AGG A048C TGT E113S TCT H179N AAT R246D GAT S312K AAGG378S TCT A048E GAG E113T ACT H179P CCT R246E GAG S312L CTG G378T ACTA048F TTT E113V GTT H179R AGG R246G GGG S312M ATG G378V GTG A048G GGTE113W TGG H179S AGT R246H CAT S312N AAT G378W TGG A048H CAT E113Y CATH179T ACT R246I ATT S312P CCT G378Y TAT A048I ATT E114A GCT H179V GTGR246K AAG S312Q CAG K379A GCT A048K AAG E114C TGT H179W TGG R246L TTGS312R CGG K379C TGT A048L CTG E114D GAT L180A GCT R246M ATG S312T ACTK379E GAG A048M ATG E114G GGG L180C TGT R246P CCT S312V GTT K379F TTTA048N AAT E114H CAT L180E GAG R246S AGT S312W TGG K379G GGG A048P CCTE114I ATT L180F TTT R246T ACG M313A GCT K379H CAT A048Q CAG E114L CTGL180G GGT R246V GTT M313C TGT K379I ATT A048R CGG E114M ATG L180H CATR246W TGG M313D GAT K379L CTT A048S TCT E114P CCT L180I ATT V247A GCGM313E GAG K379M ATG A048V GTT E114R CGG L180K AAG V247C TGT M313F TTTK379N AAT A048W TGG E114S TCT L180M ATG V247F TTT M313G GGG K379R CGTA048Y TAT E114T ACT L180N AAT V247H CAT M313H CAT K379S TCT T049A GCGE114V GTG L180P CCT V247I ATT M313K AAG K379T ACT T049C TGT E114W TGGL180R AGG V247L CTG M313L CTT K379V GTT T049D GAT E114Y TAT L180S TCGV247M ATG M313P CCT K379W TGG T049F TTT W115A GCT L180T ACT V247N AATM313R CGT F380A GCT T049G GGG W115C TGT L180W TGG V247P CCT M313S TCGF380C TGT T049H CAT W115D GAT W181A GCT V247Q CAG M313T ACT F380D GATT049I ATT W115F TTT W181C TGT V247R CGT M313V GTT F380E GAG T049K AAGW115G GGT W181D GAT V247S TCT M313Y TAT F380G GGG T049L TTG W115H CATW181E GAG V247T ACT K314A GCT F380I ATT T049N AAT W115I ATT W181F TTTV247W TGG K314C TGT F380L CTT T049P CCG W115K AAG W181H CAT V247Y TATK314D GAT F380P CCT T049R AGG W115L CTT W181I ATT R248A GCT K314H CATF380Q CAG T049S TCG W115M ATG W181K AAG R248C TGT K314I ATT F380R CGGT049V GTT W115P CCT W181L CTG R248D GAT K314L TTG F380S AGT T049W TGGW115R CGG W181M ATG R248E GAG K314N AAT F380T ACT G050A GCG W115S AGTW181N AAT R248G GGG K314P CCT F380V GTG G050C TGT W115V GTG W181Q CAGR248H CAT K314Q CAG F380W TGG G050D GAT W115Y TAT W181R CGT R248I ATTK314R CGG F380Y TAT G050E GAG R116A GCT W181S TCT R248L CTT K314S TCGT381A AGC G050F TTT R116C TGT W181V GTG R248M ATG K314T ACT T381E GAGG050H CAT R116D GAT G182A GCT R248P CCG K314V GTT T381F TTT G050L CTTR116E GAG G182C TGT R248S TCG K314W TGG T381G GGT G050M ATG R116G GGGG182D GAT R248T ACG K314Y TAT T381H CAT G050P CCT R116H CAT G182E GAGR248V GTG S315A GCT T381K AAG G050Q CAG R116I ATT G182H CAT R248W TGGS315C TGT T381L TTG G050R CGG R116L CTG G182L CTT R248Y TAT S315E GAGT381N AAT G050S AGT R116N AAT G182M ATG E249A GCT S315G GGT T381P CCTG050V GTT R116P CCT G182N AAT E249G GGG S315H CAT T381Q CAG G050W TGGR116Q CAG G182P CCT E249H CAT S315I ATT T381R CGT G050Y TAT R116S TCTG182Q CAG E249I ATT S315K AAG T381S AGT Q051A GCG R116T ACT G182R CGTE249K AAG S315L CTG T381V GTG Q051C TGT R116V GTG G182S AGT E249L CTGS315M ATG T381W TGG Q051D GAT R116W TGG G182T ACT E249M ATG S315P CCTT381Y TAT Q051F TTT P117D GAT G182V GTT E249P CCT S315R CGG V382E GAGQ051H CAT P117E GAG G182Y TAT E249Q CAG S315T ACT V382G GGG Q051I ATTP117F TTT Y183A GCT E249R CGG S315V GTT V382H CAT Q051K AAG P117G GGTY183C TGT E249S TCT S315W TGG V382I ATT Q051M ATG P117H CAT Y183D GATE249T ACT S315Y TAT V382K AAG Q051N AAT P117I ATT Y183E GAG E249V GTGC316A GCT V382L TTG Q051P CCT P117K AAG Y183G GGG E249W TGG C316D GATV382M ATG Q051R CGG P117N AAT Y183I ATT E249Y TAT C316E GAG V382N AATQ051S TCT P117Q CAG Y183K AAG A250C TGT C316G GGG V382P CCT Q051T ACGP117R AGG Y183L TTG A250F TTT C316I ATT V382Q CAG Q051W TGG P117S TCGY183N AAT A250G GGT C316K AAG V382R CGG Q051Y TAT P117T ACT Y183P CCTA250H CAT C316L CTG V382S TCG G052A GCT P117V GTT Y183Q CAG A250K AAGC316M ATG V382T ACT G052C TGT P117W TGG Y183R CGT A250L CTG C316P CCTV382W TGG G052E GAG P117Y TAT Y183S TCT A250M ATG C316R AGG V382Y TATG052F TTT T118C TGT Y183V GTT A250N AAT C316S TCT R383A GCT G052H CATT118D GAT Y183W TGG A250P CCT C316T ACT R383E GAG G052K AAG T118E GAGY184A GCT A250Q CAG C316V GTT R383F TTT G052L CTT T118G GGG Y184C TGTA250R AGG C316W TGG R383G GGG G052N AAT T118H CAT Y184D GAT A250S TCTC316Y TAT R383H CAT G052P CCT T118K AAG Y184E GAG A250T ACG L317A GCTR383I ATT G052Q CAG T118L CTG Y184F TTT A250V GTG L317C TGT R383K AAGG052R CGG T118M ATG Y184G GGT A250W TGG L317D GAT R383L CTG G052S AGTT118N AAT Y184H CAT I251C TGT L317G GGG R383M ATG G052T ACT T118P CCTY184K AAG I251D GAT L317H CAT R383N AAT G052W TGG T118Q CAG Y184L CTTI251F TTT L317I ATT R383P CCT G052Y TAT T118R CGT Y184M ATG I251G GGGL317K AAG R383S TCG V053A GCG T118V GTT Y184P CCT I251H CAT L317M ATGR383T ACT V053C TGT T118W TGG Y184R AGG I251K AAG L317N AAT R383V GTGV053D GAT T118Y TAT Y184S TCG I251L CTT L317P CCT R383W TGG V053E GAGW119A GCT Y184V GTG I251M ATG L317Q CAG G384A GCT V053G GGG W119D GATY184W TGG I251P CCG L317R AGG G384C TGT V053H CAT W119E GAG L185A GCTI251Q CAG L317S TCG G384D GAT V053L CTG W119F TTT L185D GAT I251S AGTL317T ACT G384E GAG V053N AAT W119G GGT L185E GAG I251T ACT L317W TGGG384F TTT V053P CCG W119I ATT L185F TTT I251V GTG L318C TGT G384H CATV053Q CAG W119K AAG L185G GGG I251W TGG L318D GAT G384I ATT V053R CGGW119L CTG L185I ATT I251Y TAT L318F TTT G384K AAG V053S AGT W119N AATL185K AAG R252A GCT L318G GGG G384L CTT V053T ACT W119P CCT L185N AATR252D GAT L318H CAT G384M ATG V053W TGG W119Q CAG L185P CCT R252E GAGL318I ATT G384P CCT V053Y TAT W119R CGG L185R CGG R252F TTT L318K AAGG384Q CAG T054A GCG W119S TCT L185S TCG R252G GGT L318M ATG G384R AGGT054D GAT W119V GTT L185T ACT R252H CAT L318N AAT G384S TCG T054E GAGW119Y TAT L185V GTG R252I ATT L318P CCT G384T ACT T054F TTT A120C TGTL185W TGG R252K AAG L318Q CAG K385A GCT T054G GGG A120D GAT L185Y TATR252L CTG L318R CGG K385C TGT T054H CAT A120F TTT F186A GCT R252N AATL318S AGT K385G GGG T054I ATT A120G GGG F186D GAT R252P CCT L318T ACTK385H CAT T054M ATG A120H CAT F186G GGT R252S TCG L318W TGG K385L CTTT054N AAT A120I ATT F186H CAT R252T ACT L319C TGT K385M ATG T054P CCGA120L CTT F186I ATT R252V GTG L319E GAG K385N AAT T054Q CAG A120N AATF186K AAG R252Y TAT L319F TTT K385P CCG T054R CGT A120P CCT F186L CTTV253A GCG L319G GGG K385Q CAG T054S AGT A120R CGT F186N AAT V253D GATL319H CAT K385R CGT T054V GTT A120S TCT F186P CCT V253E GAG L319I ATTK385S TCT T054Y TAT A120T ACT F186Q CAG V253G GGG L319K AAG K385T ACGI055A GCT A120V GTG F186R AGG V253H CAT L319M ATG K385V GTT I055C TGTA120W TGG F186S TCT V253I ATT L319P CCT K385W TGG I055D GAT A120Y TATF186V GTT V253L CTG L319Q CAG K385Y TAT I055F TTT R121A GCT F186W TGGV253M ATG L319R AGG P386A GCG I055G GGG R121C TGT F186Y TAT V253N AATL319S TCG P386C TGT I055H CAT R121D GAT P187A GCT V253P CCT L319V GTTP386F TTT I055L CTG R121E GAG P187F TTT V253Q CAG L319W TGG P386G GGGI055N AAT R121F TTT P187G GGG V253R CGG L319Y TAT P386H CAT I055P CCTR121G GGT P187H CAT V253S TCG D320C TGT P386I ATT I055Q CAG R121H CATP187I ATT V253T ACG D320E GAG P386L CTT I055R CGT R121K AAG P187L CTTV253W TGG D320F TTT P386M ATG I055S TCG R121L CTG P187M ATG S254C TGTD320G GGG P386N AAT I055T ACT R121M ATG P187N AAT S254D GAT D320H CATP386Q CAG I055V GTT R121P CCT P187Q CAG S254E GAG D320I ATT P386R CGTI055Y TAT R121S TCG P187R AGG S254G GGG D320K AAG P386S AGT F056A GCGR121T ACT P187S TCG S254I ATT D320L TTG P386T ACG F056C TGT R121V GTTP187T ACT S254K AAG D320M ATG P386V GTT F056E GAG R121W TGG P187V GTTS254L TTG D320N AAT P386Y TAT F056G GGG R121Y TAT P187W TGG S254N AATD320P CCT T387C TGT F056H CAT N122A GCT P187Y TAT S254P CCT D320R AGGT387E GAG F056I ATT N122C TGT D188A GCT S254Q CAG D320S AGT T387F TTTF056K AAG N122E GAG D188C TGT S254R CGG D320V GTG T387G GGG F056L TTGN122F TTT D188F TTT S254T ACT D320W TGG T387H CAT F056N AAT N122I ATTD188G GGG S254V GTG D320Y TAT T387I ATT F056P CCG N122K AAG D188H CATS254W TGG N321A GCT T387K AAG F056R CGT N122L CTG D188L CTT S254Y TATN321D GAT T387L CTG F056S TCT N122M ATG D188M ATG K255A GCG N321E GAGT387M ATG F056T ACT N122P CCT D188N AAT K255C TGT N321G GGT T387N AATF056V GTT N122Q CAG D188P CCT K255D GAT N321H CAT T387Q CAG F056W TGGN122R CGG D188Q CAG K255G GGT N321I ATT T387S TCG Y057A GCT N122S TCTD188R AGG K255H CAT N321K AAG T387V GTT Y057D GAT N122T ACT D188S AGTK255L TTG N321L CTG T387W TGG Y057E GAG N122V GTT D188T ACT K255N AATN321M ATG T387Y TAT Y057F TTT N122W TGG D188V GTG K255P CCG N321P CCTL388A GCG Y057G GGG W123A GCT D188W TGG K255Q CAG N321R CGG L388C TGTY057I ATT W123C TGT C189A GCT K255R CGG N321S TCT L388F TTT Y057L TTGW123D GAT C189E GAG K255S TCG N321T ACT L388G GGG Y057M ATG W123E GAGC189G GGT K255T ACT N321V GTG L388H CAT Y057P CCG W123G GGG C189H CATK255V GTT N321Y TAT L388I ATT Y057Q CAG W123H CAT C189K AAG K255W TGGY322C TGT L388M ATG Y057R CGG W123L CTT C189L TTG K255Y TAT Y322D GATL388P CCT Y057S AGT W123M ATG C189M ATG I256A GCT Y322E GAG L388Q CAGY057T ACG W123P CCT C189N ACT I256C TGT Y322F TTT L388R CGT Y057V GTGW123Q CAG C189P CCT I256D GAT Y322G GGT L388S TCG Y057W TGG W123R AGGC189R AGG I256E GAG Y322H CAT L388T ACG V058A GCT W123S AGT C189S TCGI256G GGG Y322I ATT L388V GTT V058C TGT W123T ACT C189T ACT I256H CATY322L CTG L388W TGG V058D GAT W123V GTT C189V GTG I256L CTT Y322N AATL388Y TAT V058G GGT W123Y TAT C189W TGG I256M ATG Y322P CCT E389A GCTV058H CAT K124A GCT C189Y TAT I256N AAT Y322R CGT E389F TTT V058I ATTK124C TGT Y190C TGT I256P CCG Y322S TCT E389G GGT V058K AAG K124D GATY190E GAG I256Q CAG Y322T ACT E389H CAT V058L CTT K124E GAG Y190F TTTI256R AGG Y322V GTG E389I ATT V058N AAT K124F TTT Y190G GGG I256T ACGY322W TGG E389K AAG V058P CCT K124G GGG Y190H CAT I256V GTT M323A GCTE389L CTG V058Q CAG K124H CAT Y190K AAG I256W TGG M323C TGT E389M ATGV058R CGG K124I ATT Y190L CTT P257A GCG M323E GAG E389P CCT V058S TCGK124L CTT Y190N AAT P257C TGT M323F TTT E389Q CAG V058W TGG K124N AATY190P CCT P257D GAT M323G GGG E389R CGG V058Y TAT K124P CCT Y190Q CAGP257G GGG M323H CAT E389S TCG D059A GCT K124R CGG Y190R CGT P257I ATTM323I ATT E389T ACT D059E GAG K124S TCT Y190S TCT P257K AAG M323K AAGE389V GTT D059G GGG K124T ACT Y190T ACT P257L CTT M323L TTG E389Y TATD059H CAT K124V GTG Y190V GTG P257M ATG M323N AAT D390A GCG D059I ATTK124W TGG Y190W TGG P257N AAT M323P CCT D390C TGT D059L CTT P125A GCTN191A GCT P257Q CAG M323R CGG D390E GAG D059M ATG P125C TGT N191E GAGP257R CGT M323S AGT D390F TTT D059N AAT P125D GAT N191F TTT P257S TCGM323T ACT D390G GGG D059P CCT P125G GGG N191G GGG P257T ACG M323V GTTD390H CAT D059Q CAG P125H CAT N191K AAG P257V GTG E324A GCT D390L CTTD059R CGT P125I ATT N191L TTG P257W TGG E324C TGT D390N AAT D059T ACGP125L CTT N191M ATG D258A GCG E324D GAT D390P CCG D059V GTG P125N AATN191P CCT D258E GAG E324F TTT D390R CGG D059W TGG P125Q CAG N191Q CAGD258G GGG E324G GGG D390S AGT D059Y TAT P125R CGT N191R CGG D258H CATE324H CAT D390T ACT R060A GCG P125S TCG N191S TCG D258I ATT E324L TTGD390V GTG R060D GAT P125T ACT N191T ACT D258L CTT E324M ATG D390W TGGR060F TTT P125V GTG N191V GTT D258N AAT E324N AAT D390Y TAT R060G GGTP125W TGG N191W TGG D258P CCG E324P CCT L391A GCT R060H CAT P125Y TATN191Y TAT D258Q CAG E324R CGG L391C TGT R060I ATT K126A GCT H192C TGTD258R CGT E324S AGT L391D GAT R060K AAG K126D GAT H192F TTT D258S AGTE324V GTG L391G GGG R060L CTT K126E GAG H192G GGT D258T ACG E324W TGGL391H CAT R060N AAT K126F TTT H192K AAG D258V GTG E324Y TAT L391K AAGR060P CCG K126G GGT H192L CTT D258W TGG T325A GCT L391N AAT R060Q CAGK126H CAT H192M ATG D258Y TAT T325C TGT L391P CCT R060S TCG K126I ATTH192N AAT A259E GAG T325D GAT L391Q CAG R060T ACG K126L CTG H192P CCTA259G GGG T325E GAG L391R CGG R060V GTT K126M ATG H192Q CAG A259I ATTT325G GGT L391S TCT R060Y TAT K126N AAT H192R CGT A259K AAG T325H CATL391T ACT L061A GCT K126P CCT H192S TCG A259L TTG T325I ATT L391V GTGL061E GAG K126Q CAG H192T ACT A259M ATG T325K AAG L391W TGG L061F TTTK126R AGG H192V GTT A259N AAT T325M ATG L391Y TAT L061G GGG K126S TCTH192W TGG A259P CCT T325N AAT E392A GCT L061H CAT K126T ACT H192Y TATA259Q CAG T325Q CAG E392C TGT L061I ATT K126V GTG H193A GCT A259R CGTT325R CGG E392F TTT L061M ATG K126W TGG H193C TGT A259S AGT T325S TCGE392G GGG L061N AAT K126Y TAT H193D GAT A259T ACT T325V GTG E392K AAGL061P CCT D127A GCT H193F TTT A259V GTG T325W TGG E392L CTG L061Q CAGD127E GAG H193G GGG A259W TGG I326A GCT E392M ATG L061R AGG D127F TTTH193K AAG A259Y TAT I326C TGT E392P CCT L061T ACT D127G GGT H193L TTGK260A GCG I326D GAT E392Q CAG L061V GTT D127H CAT H193M ATG K260C TGTI326E GAG E392R AGG L061W TGG D127K AAG H193P CCG K260D GAT I326G GGGE392S AGT L061Y TAT D127L CTG H193Q CAG K260E GAG I326H CAT E392T ACTG062A GCG D127M ATG H193R AGG K260G GGG I326K AAG E392V GTT G062C TGTD127N AAT H193S TCT K260H CAT I326L CTT E392W TGG G062D GAT D127Q CAGH193T ACG K260L TTG I326N AAT E392Y TAT G062F TTT D127R CGT H193V GTGK260M ATG I326P CCT Q393A GCG G062I ATT D127S AGT H193Y TAT K260P CCGI326R CGG Q393C TGT G062K AAG D127T ACT Y194A GCT K260Q CAG I326S TCTQ393D GAT G062L CTT D127V GTT Y194C TGT K260R CGG I326V GTG Q393F TTTG062M ATG D127W TGG Y194E GAG K260S TCT I326W TGG Q393G GGT G062P CCTV128A GCT Y194F TTT K260V GTT I326Y TAT Q393H CAT G062Q CAG V128C TGTY194G GGG K260W TGG L327A GCT Q393I ATT G062R CGT V128E GAG Y194I ATTK260Y TAT L327D GAT Q393K AAG G062S AGT V128F TTT Y194L TTG S261A GCGL327E GAG Q393L TTG G062T ACT V128G GGG Y194N AAT S261E GAG L327F TTTQ393M ATG G062V GTG V128H CAT Y194P CCT S261F TTT L327G GGG Q393N AATG062Y TAT V128I ATT Y194Q CAG S261G GGG L327H CAT Q393P CCG Y063A GCGV128K AAG Y194R AGG S261I ATT L327M ATG Q393R CGT Y063C TGT V128L CTGY194S TCG S261K AAG L327N AAT Q393S TCG Y063G GGT V128P CCT Y194T ACGS261L CTT L327Q CAG Q393T ACG Y063H CAT V128Q CAG Y194V GTG S261M ATGL327R CGG F394A GCG Y063I ATT V128R AGG Y194W TGG S261N AAT L327S AGTF394D GAT Y063K AAG V128S TCG K195A GCG S261P CCT L327T ACT F394E GAGY063L CTG V128W TGG K195E GAG S261Q CAG L327V GTG F394G GGG Y063M ATGV128Y TAT K195F TTT S261R CGT L327W TGG F394I ATT Y063N AAT Y129A GCTK195G GGT S261T ACT L327Y TAT F394K AAG Y063P CCT Y129C TGT K195H CATS261V GTT N328A GCT F394L CTG Y063R AGG Y129D GAT K195I ATT S261W TGGN328C TGT F394N AAT Y063S TCT Y129E GAG K195L TTG P262A GCG N328D GATF394P CCG Y063T ACG Y129G GGG K195N AAT P262D GAT N328G GGT F394Q CAGY063V GTG Y129H CAT K195Q CAG P262E GAG N328H CAT F394R CGT Y063W TGGY129L TTG K195R CGT P262F TTT N328I ATT F394S TCG Y064A GCT Y129M ATGK195S TCT P262G GGG N328K AAG F394T ACT Y064C TGT Y129P CCT K195T ACTP262H CAT N328L CTT F394V GTT Y064D GAT Y129Q CAG K195V GTG P262I ATTN328Q CAG F394W TGG Y064E GAG Y129R CGG K195W TGG P262K AAG N328R AGGS395A GCG Y064F TTT Y129S AGT K195Y TAT P262Q CAG N328S AGT S395C TGTY064G GGT Y129T ACT K196A GCT P262R CGT N328T ACT S395D GAT Y064H CATY129V GTT K196C TGT P262S TCT N328V GTG S395E GAG Y064I ATT Y129W TGGK196D GAT P262T ACT N328W TGG S395G GGG Y064K AAG K130C TGT K196E GAGP262V GTG N328Y TAT S395H CAT Y064L CTT K130D GAT K196G GGG P262W TGGP329C TGT S395K AAG Y064P CCT K130E GAG K196I ATT P262Y TAT P329F TTTS395L CTT Y064Q CAG K130G GGG K196L TTG L263A GCT P329G GGT S395M ATGY064R CGG K130H CAT K196N AAT L263E GAG P329H CAT S395P CCT Y064S AGTK130I ATT K196P CCG L263F TTT P329I ATT S395R CGG Y064T ACT K130L TTGK196R CGT L263G GGG P329K AAG S395T ACG Y064V GTT K130N AAT K196S TCGL263H CAT P329L CTG S395V GTT Y064W TGG K130Q CAG K196T ACT L263K AAGP329N AAT S395W TGG P065A GCT K130R AGG K196V GTG L263M ATG P329Q CAGS395Y TAT P065C TGT K130S TCT K196W TGG L263N AAT P329R CGT E396A GCGP065D GAT K130T ACT K196Y TAT L263P CCG P329S AGT E396C TGT P065F TTTK130V GTG P197A GCT L263Q CAG P329T ACT E396D GAT P065G GGG K130W TGGP197C TGT L263R CGG P329V GTT E396F TTT P065H CAT K130Y TAT P197D GATL263S AGT P329W TGG E396G GGG P065I ATT N131C TGT P197E GAG L263T ACTP329Y TAT E396H CAT P065K AAG N131E GAG P197F TTT L263V GTT Y330A GCTE396I ATT P065N AAT N131F TTT P197G GGT L263W TGG Y330C TGT E396L CTTP065R CGG N131G GGG P197H CAT P264A GCG Y330D GAT E396P CCG P065S TCGN131H CAT P197K AAG P264D GAT Y330E GAG E396Q CAG P065T ACG N131I ATTP197L TTG P264E GAG Y330F TTT E396R AGG P065V GTT N131L CTT P197M ATGP264F TTT Y330G GGT E396S TCT P065W TGG N131M ATG P197Q CAG P264G GGTY330I ATT E396T ACT P065Y TAT N131P CCT P197R CGT P264H CAT Y330L CTGE396V GTG Y066A GCG N131Q CAG P197S AGT P264L CTT Y330M ATG E396Y TATY066C TGT N131R CGG P197T ACT P264M ATG Y330N AAT K397A GCT Y066D GATN131S AGT P197W TGG P264N AAT Y330P CCT K397C TGT Y066E GAG N131T ACTG198A GCT P264R CGG Y330R AGG K397E GAG Y066G GGT N131V GTG G198C TGTP264S AGT Y330S AGT K397F TTT Y066H CAT N131Y TAT G198D GAT P264T ACTY330V GTT K397G GGT Y066I ATT R132A GCT G198E GAG P264V GTT I331V GTGK397I ATT Y066K AAG R132C TGT G198H CAT P264W TGG Y330W TGG K397L TTGY066L CTG R132E GAG G198L CTG P264Y TAT I331A GCT K397M ATG Y066N AATR132F TTT G198N AAT V265A GCG I331C TGT K397N AAT Y066P CCT R132H CATG198P CCG V265C TGT I331D GAT K397P CCG Y066R CGG R132I ATT G198Q CAGV265D GAT I331E GAG K397Q CAG K397T ACT R132K AAG G198R AGG V265E GAGI331F TTT K397R AGG K397V GTT R132L TTG G198S TCT V265F TTT I331H CATK397S TCG F398A GCT L406P CCT K415G GGT C423T ACT A432L TTG E441D GATF398C TGT L406Q CAG K415L CTG C423V GTG A432M ATG E441F TTT F398E GAGL406R CGG K415M ATG C423W TGG A432N AAT E441G GGG F398G GGT L406S AGTK415P CCG I424A GCT A432P CCT E441H CAT F398H CAT L406T ACG K415Q CAGI424C TGT A432R AGG E441K AAG F398I ATT L406V GTT K415R CGG I424E GAGA432S TCT E441L CTT F398L CTT L406Y TAT K415S TCT I424G GGG A432V GTGE441N AAT F398N AAT S407A GCG K415T ACT I424H CAT A432Y TAT E441Q CAGF398P CCT S407D GAT K415V GTG I424K AAG F433A GCT E441R CGG F398R AGGS407E GAG K415W TGG I424L CTT F433C TGT E441S AGT F398S TCT S407F TTTK415Y TAT I424N AAT F433D GAT E441T ACT F398T ACT S407G GGT D416C TGTI424Q CAG F433E GAG E441V GTG F398V GTT S407H CAT D416F TTT I424R CGGF433G GGG E441Y TAT F398W TGG S407L CTG D416G GGT I424S TCG F433H CATE442C TGT F398Y TAT S407M ATG D416H CAT I424T ACT F433I ATT E442G GGGY399A GCG S407N AAT D416I ATT I424V GTT F433K AAG E442H CAT Y399C TGTS407P CCT D416K AAG I424W TGG F433L TTG E442K AAG Y399D GAT S407Q CAGD416L CTT I424Y TAT F433P CCT E442L CTT Y399E GAG S407R CGG D416N AATA425C TGT F433R CGG E442M ATG Y399G GGG S407T ACG D416Q CAG A425D GATF433S AGT E442N AAT Y399K AAG S407V GTG D416R CGG A425E GAG F433T ACTE442P CCT Y399M ATG S407W TGG D416S TCT A425G GGT F433V GTG E442Q CAGY399N AAT C408A GCG D416T ACG A425I ATT F433W TGG E442R CGG Y399P CCTC408E GAG D416V GTG A425K AAG L434F TTT E442S AGT Y399Q CAG C408F TTTD416W TGG A425L TTG L434G GGT E442T ACT Y399R CGG C408G GGG D416Y TATA425M ATG L434H CAT E442V GTG Y399S TCG C408I ATT T417A GCT A425N AATL434I ATT E442W TGG Y399T ACG C408K AAG T417D GAT A425P CCT L434K AAGE442Y TAT Y399V GTT C408L CTT T417E GAG A425R AGG L434M ATG P443A GCTY399W TGG C408N AAT T417F TTT A425S AGT L434N AAT P443D GAT C400A GCGC408P CCT T417G GGG A425V GTG L434P CCT P443E GAG C400D GAT C408R CGTT417H CAT A425W TGG L434Q CAG P443F TTT C400E GAG C408S TCG T417I ATTA425Y TAT L434R CGG P443G GGG C400F TTT C408T ACT T417K AAG D426A GCTL434S AGT P443H CAT C400G GGG C408V GTT T417L TTG D426C TGT L434T ACTP443I ATT C400I ATT C408W TGG T417M ATG D426E GAG L434V GTT P443L CTTC400L CTG C408Y TAT T417P CCT D426F TTT L434W TGG P443M ATG C400M ATGK409A GCG T417Q CAG D426G GGG L434Y TAT P443N AAT C400P CCG K409C TGTT417R CGT D426I ATT K435A GCT P443Q CAG C400Q CAG K409D GAT T417S TCGD426K AAG K435C TGT P443R AGG C400R CGG K409E GAG T417W TGG D426L CTGK435E GAG P443S TCT C400S AGT K409G GGT D418A GCT D426M ATG K435F TTTP443T ACT C400T ACG K409H CAT D418C TGT D426N AAT K435G GGT P443W TGGC400V GTG K409I ATT D418E GAG D426P CCT K435H CAT Q444C TGT C400Y TATK409L CTG D418F TTT D426Q CAG K435I ATT Q444D GAT S401A GCT K409P CCGD418G GGT D426R CGT K435L CTG Q444E GAG S401C TGT K409Q CAG D418I ATTD426S TCG K435P CCT Q444F TTT S401D GAT K409R AGG D418L TTG D426Y TATK435R AGG Q444G GGG S401E GAG K409S TCG D418M ATG G427A GCT K435S TCTQ444H CAT S401F TTT K409T ACG D418N AAT G427C TGT K435T ACT Q444I ATTS401G GGG K409V GTG D418P CCT G427F TTT K435V GTT Q444K AAG S401H CATK409W TGG D418Q CAG G427H CAT K435W TGG Q444L CTG S401K AAG A412Y TATD418R CGG G427I ATT K435Y TAT Q444M ATG S401L CTT E410D GAT D418S TCGG427K AAG P436C TGT Q444N AAT S401N AAT E410G GGG D418V GTG G427L CTGP436D GAT Q444R CGG S401Q CAG E410I ATT D418Y TAT G427P CCT P436E GAGQ444V GTT S401R CGT E410K AAG A419D GAT G427Q CAG P436G GGG Q444W TGGS401T ACT E410L CTT A419E GAG G427R CGT P436H CAT Q444Y TAT S401W TGGE410M ATG A419F TTT G427S AGT P436I ATT I445A GCT S401Y TAT E410N AATA419G GGG G427T ACT P436K AAG I445C TGT C402A GCT E410P CCG A419H CATG427V GTG P436L CTG I445D GAT C402D GAT E410Q CAG A419I ATT G427W TGGP436M ATG I445G GGG C402E GAG E410R CGT A419K AAG G427Y TAT P436Q CAGI445H CAT C402F TTT E410S TCG A419L CTT V428A GCT P436R CGG I445K AAGC402G GGG E410T ACG A419N AAT V428C TGT P436S TCT I445L CTT C402L TTGE410V GTG A419P CCT V428D GAT P436T ACT I445M ATG C402M ATG E410W TGGA419R CGG V428E GAG P436W TGG I445N AAT C402P CCT E410Y TAT A419S TCTV428F TTT P436Y TAT I445P CCT C402Q CAG K411A GCT A419T ACT V428G GGTP437A GCT I445Q CAG C402R CGG K411D GAT A419W TGG V428H CAT P437D GATI445R AGG C402S TCT K411E GAG A419Y TAT V428L CTT P437F TTT I445S AGTC402T ACG K411F TTT V420A GCT V428M ATG P437G GGT I445T ACT C402V GTTK411G GGG V420D GAT V428N AAT P437H CAT I445V GTG C402W TGG K411H CATV420F TTT V428P CCT P437I ATT I445W TGG C402Y TAT K411I ATT V420G GGTV428R CGG P437K AAG I445Y TAT Y403A GCT K411L CTG V420H CAT V428S TCGP437L CTG F446A GCT Y403C TGT K411N AAT V420I ATT V428T ACT P437M ATGF446C TGT Y403E GAG K411P CCT V420K AAG V428Y TAT P437Q CAG F446D GATY403F TTT K411R AGG V420L CTT C429A GCT P437R CGT F446E GAG Y403G GGTK411S TCG V420N AAT C429D GAT P437S TCT F446G GGG Y403H CAT K411T ACTV420P CCT C429G GGT P437T ACT F446H CAT Y403K AAG K411V GTT V420R AGGC429I ATT P437W TGG F446I ATT Y403L TTG K411W TGG V420S TCT C429K AAGP437Y TAT F446K AAG Y403M ATG A412D GAT V420T ACT C429L TTG M438A GCTF446L TTG Y403N AAT A412E GAG V420W TGG C429M ATG M438C TGT F446M ATGY403P CCG A412G GGG V420Y TAT C429N AAT M438D GAT F446Q CAG Y403Q CAGA412H CAT D421A GCT C429P CCT M438E GAG F446R CGG Y403R CGG A412I ATTD421E GAG C429R CGG M438G GGG F446T ACT Y403S TCT A412L CTG D421G GGTC429S TCG M438L TTG F446V GTT Y403T ACG A412N AAT D421H CAT C429T ACTM438N AAT F446W TGG S404A GCT A412P CCT D421I ATT C429V GTT M438P CCTY447D GAT S404C TGT A412Q CAG D421K AAG C429W TGG M438Q CAG Y447E GAGS404D GAT A412R CGG D421L TTG C429Y TAT M438R AGG Y447F TTT S404F TTTA412S AGT D421M ATG I430A GCT M438S TCG Y447G GGT S404G GGT A412V GTTD421N AAT I430D GAT M438T ACT Y447I ATT S404H CAT A412W TGG D421Q CAGI430E GAG M438V GTG Y447K AAG S404L CTT D413A GCG D421R CGG I430G GGGM438W TGG Y447L CTT S404M ATG D413E GAG D421S TCG I430H CAT M438Y TATY447M ATG S404N AAT D413F TTT D421T ACT I430K AAG E439A GCT Y447N AATS404P CCT D413G GGT D421W TGG I430L TTG E439C TGT Y447P CCT S404R AGGD413H CAT D421Y TAT I430M ATG E439F TTT Y447Q CAG S404T ACG D413I ATTV422A GCT I430N AAT E439G GGG Y447R AGG S404V GTG D413K AAG V422C TGTI430P CCT E439H CAT Y447T ACT S404W TGG D413L CTG V422D GAT I430R AGGE439K AAG Y447V GTT S404Y TAT D413N AAT V422E GAG I430S TCT E439L CTTY447W TGG T405A GCG D413P CCG V422G GGG I430T ACT E439N AAT T405C TGTD413Q CAG V422H CAT I430V GTT E439P CCT T405F TTT D413R CGT V422I ATTI430W TGG E439Q CAG T405G GGG D413S TCG V422L CTG D431A GCT E439R CGGT405I ATT D413T ACT V422M ATG D431E GAG E439S TCG T405K AAG D413W TGGV422N AAT D431G GGT E439T ACT T405L TTG V414A GCG V422P CCT D431H CATE439V GTT T405M ATG V414D GAT V422Q CAG D431I ATT E439W TGG T405P CCGV414E GAG V422R CGT D431K AAG T440A GCT T405Q CAG V414F TTT V422S TCGD431L CTT T440D GAT T405R CGT V414G GGT V422T ACT D431N AAT T440E GAGT405S TCT V414H CAT V422W TGG D431P CCT T440F TTT T405V GTG V414I ATTV422Y TAT D431Q CAG T440G GGG T405W TGG V414K AAG C423A GCT D431R CGTT440H CAT T405Y TAT V414L TTG C423D GAT D431S TCT T440I ATT L406A GCTV414M ATG C423E GAG D431V GTT T440L CTT L406C TGT V414Q CAG C423F TTTD431W TGG T440M ATG L406D GAT V414R AGG C423G GGG D431Y TAT T440P CCTL406E GAG V414S TCG C423H CAT A432C TGT T440Q CAG L406F TTT V414T ACTC423L CTG A432E GAG T440R AGG L406G GGT V414Y TAT C423M ATG A432F TTTT440S AGT L406I ATT K415A GCG C423P CCT A432G GGG T440V GTG L406N AATK415C TGT C423Q CAG A432H CAT T440Y TAT K415D GAT C423R AGG A432I ATTE441A GCT K415E GAG C423S TCG A432K AAG E441C TGT

2. Expression

For expression of each mutant, HZ24-PH20-IRES-SEAP plasmid DNAcontaining cDNA encoding one of the variant PH20 or encoding wildtypePH20 was transfected into monolayer CHO-S cells (Invitrogen, Cat. No.11619-012) using Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027)according to the protocol suggested by the manufacturer. CHO-S cellswere seeded the night before transfection and grown in DMEM with 10% FBSto be 80% confluent the next day. Then, the medium of the CHO-S cellswas replaced with Opti-MEM. A mixture of plasmid DNA and lipofectaminewas made (0.2 μg DNA and 0.5 μL Lipofetamine). The Lipofectamine/DNAmixture was added to CHO-S cells and incubated overnight. The next day,the cells were supplemented with CD-CHO serum free medium (Invitrogen,Cat. No. 10743-029). Supernatant from transfected cells was collected atvarious time points after transfection, and generally 96 hours aftertransfection. The supernatant, containing the variant PH20 protein orwildtype PH20 having a sequence of amino acids set forth in SEQ ID NO:3,was stored at −20° C. Activities of the supernatants were screened asdescribed in the following examples.

Example 3 Screening of Library with a Hyaluronidase Activity Assay toIdentify Activity Mutants

In this example, supernatants of expressed PH20 variants generated inExample 2 were screened using a hyaluronidase activity assay to assessactivity of each mutant. In addition, activity of the secreted alkalinephosphatase (SEAP) was also measured to allow for normalizing PH20activity of the expressed mutants to the PH20 wildtype. Active andinactive mutants were identified.

1. Generation of Biotinylated HA (bHA) Substrate

A 1.2-MDa HA (Lifecore) was biotinylated for use as a substrate in thehyaluronidase activity assay. First, 1.2 grams (g) of 1.2 MDa HA wasdissolved at 4° C. in 600 mL ddH₂0 for a week at a concentration of 2mg/mL with stirring. Next, 645.71 mg Biotin Hydrazide was dissolved in100 mL DMSO to a concentration of 25 mM (6.458 mg/mL, 247.8 mg in 38.37mL DMSO). The biotin solution was warmed briefly at 37° C. until thesolution was clear. Also, 368.61 mg Sulfo-NHS in 20 mL ddH₂O wasdissolved to make a 100× solution (18.4 mg/mL Sulfo-NHS). A 30 mM(1000×) water-soluble carbodiimide EDC solution was made by dissolving17.63 mg EDC in 3 mL ddH20 at a concentration of 5.7513 mg/mL rightbefore the reaction was started.

To four (4) 1000-mL sterile capped bottles, the following componentswere added at room temperature (RT) and in the following order withstirring: 1) 200 mL of 2 mg/mL HA solution; 2) 80 mL of 0.5M MES, pH 5.0with gentle mixing; and 3) 91.6 mL of ddH₂O with gentle mixing. Next, 24mL of 25 mM Biotin-Hydrazide and 4 mL of 100× Sulfo-NHS solution wereadded sequentially, immediately followed by the addition of 500 μL EDC.After the addition of each component, the solution was mixed byinverting three times and stirring. After the addition of the lastcomponent, the solution was mixed by stirring overnight at 4° C. Then,Guanidine hydrochloride was added to a final concentration of 4 M byadding 38.2 g per 100 mL and was allowed to dissolve completely beforeadjusting the solution volume to 600 mL with ddH₂O.

For dialysis, 200 mL from each batch of the conjugated HA guanidinehydrochloride solution was transferred into dialysis membranes. Over thecourse of three days, the solution was dialyzed against ddH₂O with achange in ddH₂O at least six times. The resulting volume of about 840 mLwas adjusted to a final volume of 1000 mL with ddH₂O. The finalconcentration of the biotinylated hyaluronan (bHA) was 0.4 mg/mL.

2. Hyaluronidase Activity Assay

The enzyme assay was a modification of the method described by Frost etal. (1997) (A Microtiter-Based Assay for Hyaluronidase Activity NotRequiring Specialized Reagents. Analytical Biochemistry (1997)251:263-269) that provides a measure of PH20 hyaluronidase activity.

First, biotinylated HA (bHA) substrate was bound to plastic microtiterplates to generate assay plates. Briefly, 100 μl of b-HA at 1 mg/mL in0.5 M carbonate buffer (pH 9.6) was dispensed into each well of a highbind microplate (Immunolon 4 HBX extra high binding; Thermo Scientific).The plate was covered with a plate sealer and stored between 2-8° C. for24-48 hours.

Then, the assay plate was washed with 1× phosphate buffered saline (PBS)wash buffer containing 0.05% (v/v) Tween 20 (PBST). PBST was generatedfrom 1× PBS (generated from Catalog No. P5368, Sigma (10 mM PhosphateBuffer, 2.7 mM Potassium Chloride, 137 mM Sodium Chloride, pH 7.4) byplacing the contents of one packet of PBS into a 1-L graduated cylinderwith 800 mL deionized water, dissolved by stirring or shaking and addingsufficient quantity of water to 1 L) by adding 500 μl Tween 20 (CatalogNo. 6505; EMD Bioscience) to 900 mL of 1× PBS and adding sufficientquantity of water to 1 L. Washing was done using the BioTek ELx405Select CW plate washer (BioTek) by washing five (5) times with 300 μlPBST wash buffer per well for each wash. At the end of each wash, theplate was tapped on a paper towel to remove excess liquid from eachwell. Prior to incubation with samples, 200 μl Blocking Buffer (1.0% w/vBovine Serum Albumin (BSA) in PBS) was added to each well and the assayplate was incubated at 37° C. for approximately 1 hour prior. TheBlocking buffer was generated by adding 2.5 g of BSA (Catalog No.001-000-162; Jackson Immuno Research) to 200 mL 1× PBS, stirring, addinga sufficient quantity of 1× PBS to 250 mL and filtering through an 0.2μM PES filter unit.

Transfected variant or wildtype PH20 supernatants generated as describedin Example 1 were diluted in duplicate 1:25 in assay diluent buffer (pH7.4 HEPES buffer; 10 mM HEPES, 50 mM NaCl, 1 mM CaCl₂, 1 mg/mL BSA, pH7.4, 0.05% Tween-20) in uncoated 4×HB high bound microplates. For thestandard curve, 1:3 serial dilutions of rHuPH20 (generated as describedin Example 1 with a specific activity of 145 U/mL) were made in assaydiluent buffer in duplicate starting from 3 U/mL for standards asfollows: 3 U/mL, 1 U/mL, ⅓ U/mL, 1/9 U/mL, 1/27 U/mL, 1/81 U/mL, and1/243 U/mL. One hundred microliters (100 up of each standard and samplewere transferred to the assay plates and incubated for approximately 1.5hours at 37° C.

After the incubation, the plate was washed with PBST using the BioTekELx405 Select CW plate washer by washing five (5) times with 300 μl PBSTwash buffer per well for each wash. At the end of each wash, the platewas tapped on a paper towel to remove excess liquid from each well.Then, 100 μl of 1:5000 diluted Streptavidin-HRP (SA-HRP) was added toeach well of the plate and incubated at ambient temperature forapproximately 1 hour. For the dilution, a 1 mg/mL stock ofStreptavidin-HRP conjugate (Catalog No. 21126; Thermo Scientific) wasdiluted 1:5000 into dilution buffer (1 mg/mL BSA, 0.025% Tween20, 137 mMNaCl, 20 mM Tris pH 7.5). After the incubation, the plate was washedwith PBST using the BioTek ELx405 Select CW plate washer by washing five(5) times with 300 μl PBST wash buffer per well for each wash. At theend of each wash, the plate was tapped on a paper towel to remove excessliquid from each well. Then, 100 μl of TMB solution (Catalog No.52-00-03, KPL; ambient temperature and protected from light) was addedto each well for approximately five (5) minutes at room temperature oruntil an optimal color development was yielded. To stop the reaction,100 μl 1.0 N Sulfuric Acid or TMB Stop solution (Catalog No. 50-85-06)were added to each well and the plates tapped to mix. Optical densitywas measured at 450 nm within 30 minutes of adding the stop solution.Since more PH20 in a standard or sample would lead to less bHA availableto bind SA-HRP, the optical density (450 nm) value was inverselyproportional to the concentration of hyaluronidase activity in eachspecimen.

3. SEAP Activity

Activity of secreted alkaline phosphatase (SEAP) in the cell culturesupernatant also was measured using a colorimetric assay of placentalalkaline phosphatase using pNPP as a phosphatase substrate (AnaspecSensoLyte pNPP SEAP kit; Catalog No. 72144, Anaspec) according to themanufacturer's instructions. The absorbance signal was measured atoptical density (OD) of 405 nm.

The criteria for the high throughput (HTP) screening were that thetransfected supernatant resulted in a SEAP signal of ≥0.1 and the signalfor the rHuPH20 wildtype control produced a signal of ≥1 U/mL. Also, thecriteria for each screen were that the standard curves had a signal tonoise ratio (S/N) for the 0 U/mL standard versus the 3 u/mL standard at0D405 of ≥5, had less than three β) standards with a coefficient ofvariation (CV) ≥10%, and at least four (4) of the standards were in thelinear range.

Example 4 Selected pH20 Variants with Altered Hyaluronidase Activity

Each generated variant was screened for hyaluonidase activity asdescribed in Example 3. The SEAP expression was used to normalize PH20activity of each variant to the PH20 wildtype. Mutants were identifiedthat exhibited altered hyaluronidase activity compared to wildtype.

1. Active Mutants

Active mutants were selected whereby at least one duplicate sampleexhibited greater than 40% of wildtype activity when normalized to SEAPactivity. The identified active mutants are set forth in Table 9. TheTable sets forth the amino acid replacement compared to the sequence ofamino acids of PH20 set forth in SEQ ID NO:3. The amino acid sequence ofexemplary mutants also is set forth by reference to a SEQ ID NO. TheTable also sets forth the average hyaluronidase activity of testedduplicates normalized by SEAP values compared to average of wildtypePH20 activities in each plate, which were also normalized by their ownSEAP values. For example, a value of 0.40 indicates that the variantexhibits 40% of the hyaluronidase activity of wildtype PH20, a value of1 indicates that the variant exhibits a similar hyaluronidase activityof wildtype and a value of 3.00 indicates that the variant exhibits 300%of the hyaluronidase activity of wildtype PH20 or 3-fold increasedactivity compared to wildtype.

The results in Table 9 show that over 600 tested mutants exhibitactivity that is increased compared to wildtype. For example, about 536mutants exhibit 120% or greater than 120% of the hyaluronidase activityof wildtype PH20 and about 75 of the mutants exhibit 300% or greaterthan 300% of the hyaluronidase activity of wildtype PH20. In particular,the results in Table 9 show that that hyaluronidase activity compared towildtype of mutant S69A is about 22-fold; mutant S69R is about 14-fold;mutant 170A is about 27-fold; mutant 170K is about 14-fold; mutant 170Ris about 14-fold; and mutant I271L is about 10-fold.

TABLE 9 ACTIVE MUTANTS SEQ ID AvgNorm mutant NO Act. L001A 74 0.95 L001C0.89 L001E 75 0.55 L001F 0.41 L001G 76 0.62 L001H 73 1.90 L001K 77 1.39L001N 0.87 L001P 0.92 L001Q 78 3.27 L001R 79 0.72 L001S 0.74 L001T 0.99L001V 1.00 L001W 0.88 N002A 0.61 N002C 0.4 G291C 0.27 N002G 0.44 N002L0.46 N002P 0.54 N002Q 0.84 N002S 0.78 N002T 1.05 N002V 0.65 F003E 0.42F003H 0.68 F003L 0.59 F003Y 0.50 R004A 0.73 R004I 0.54 R004S 0.60 R004T0.66 R004V 1.09 A005H 0.44 P006A 80 0.78 P006H 0.58 P006K 0.80 P006L0.76 P006N 0.40 P006Q 0.89 P006R 0.56 P007M 0.57 V008I 1.17 V008L 0.53V008M 81 0.47 V008P 0.33 I009K 0.69 I009L 1.08 I009R 0.53 I009S 0.98I009V 0.84 P010D 0.62 P010E 0.66 P010G 83 0.55 P010H 84 0.43 P010N 0.55P010Q 0.89 P010R 0.73 P010S 0.55 P010W 0.59 N011D 0.54 N011G 0.45 N011H0.69 N011K 0.58 N011S 85 0.39 M310F 0.30 V012A 0.56 V012E 86 1.86 V012I87 0.68 V012K 88 0.65 V012L 0.44 V012N 0.46 V012R 0.50 V012S 0.75 V012T89 1.50 P013H 0.46 P013S 0.68 P013T 0.90 P013Y 0.51 F014D 0.64 F014I0.42 F014M 0.47 F014V 90 0.46 L015A 0.65 L015M 92 0.45 L015V 91 2.20A020S 93 0.50 S022H 0.57 S022M 0.49 S022T 94 0.48 S022Y 0.45 E023D 0.97F024A 0.69 F024E 95 3.99 F024G 0.75 F024H 96 2.07 F024I 0.70 F024K 0.96F024L 0.62 F024M 0.85 F024N 0.60 F024R 97 1.22 F024T 1.18 F024V 1.15F024Y 0.90 L026A 98 1.30 L026E 99 3.22 L026G 0.81 L026H 0.97 L026I 0.51L026K 100 1.88 L026M 101 1.43 L026P 0.55 L026Q 102 1.44 L026R 103 1.43L026S 0.78 L026T 0.87 L026V 0.52 L026W 0.53 L026Y 0.52 G027A 0.79 G027D104 1.22 G027E 1.18 G027F 0.61 G027H 1.11 G027I 0.41 G027K 105 2.71G027L 0.76 G027P 0.46 G027Q 1.12 G027R 106 1.88 G027S 0.94 G027T 0.61G027W 0.76 K028A 0.78 K028D 0.62 K028E 0.54 K028F 0.75 K028I 0.55 K028L0.51 K028M 0.67 K028N 0.58 K028P 0.40 K028R 107 0.71 K028S 0.46 K028T0.68 K028V 0.76 K028W 0.51 F029A 0.90 F029E 108 4.03 F029G 1.05 F029H0.82 F029I 109 1.53 F029K 110 1.34 F029L 111 2.36 F029M 112 2.08 F029P113 3.79 F029R 114 1.24 F029S 115 2.21 F029T 116 0.85 F029V 117 1.65F029W 0.48 D030A 1.12 D030F 0.84 D030G 118 2.02 D030H 119 1.69 D030K 1202.63 D030L 121 1.32 D030M 122 1.85 D030P 1.19 D030Q 0.84 D030R 123 1.82D030S 124 1.62 D030T 0.57 D030V 0.46 D030W 0.62 E031A 125 2.05 E031C 1262.95 E031G 127 1.27 E031H 128 2.74 E031I 129 3.89 E031K 130 3.13 E031L131 2.62 E031P 132 1.51 E031R 133 2.27 E031S 134 1.70 E031T 135 3.96E031V 136 4.57 E031W 137 1.26 E031Y 1.13 P032A 0.92 P032C 138 0.40 P032F139 2.71 I326C 0.39 I331C 0.27 P032G 140 1.60 P032H 141 2.08 P032K 1.04P032L 0.82 P032M 0.67 P032N 0.70 P032Q 1.11 P032R 1.17 P032S 1.01 P032T0.77 P032V 0.81 P032W 0.54 P032Y 1.01 L033G 143 0.57 L033M 0.69 L033P0.87 L033Q 0.45 L033R 0.61 L033S 0.48 L033T 0.45 L033W 142 1.58 D034A0.38 D034E 0.58 D034H 0.41 D034K 0.54 D034Q 0.59 D034R 1.17 D034W 1440.46 M035F 0.87 M035H 0.60 M035L 0.52 M035T 0.83 M035Y 0.78 S036A 0.45S036D 0.32 S036G 0.64 S036H 147 0.54 S036K 0.83 S036L 0.71 S036R 1.09Q347L 0.39 V351Q 0.34 S036T 0.51 L037F 149 3.33 L037I 0.62 L037K 0.43L037M 150 1.46 L037P 0.63 L037R 0.51 L037V 0.57 F038Y 151 1.29 S039A 1521.06 S039L 153 0.80 S039N 154 2.32 S039Q 1.10 S039R 0.56 S039T 155 1.57S039Y 0.56 F040L 156 0.92 F040W 1.11 I041A 0.67 I041C 0.53 I041D 0.78I041E 0.51 I041G 0.76 I041H 0.77 I041N 0.40 I041T 157 1.47 I041V 0.73I041W 0.66 G042A 0.64 S043T 0.43 P044E 0.59 R045I 0.45 R045K 0.53 I046A1.04 I046C 0.37 I046E 0.43 I046F 0.73 I046H 0.82 I046L 158 1.08 I046M1.00 I046N 0.66 I046R 159 2.29 I046S 0.64 I046T 0.55 I046V 1.01 I046Y0.76 N047A 0.48 N047D 160 0.82 N047F 161 1.32 N047G 0.82 N047H 1.16N047K 0.67 N047M 0.77 N047Q 0.69 N047R 0.84 N047S 0.85 N047T 162 1.49N047W 163 0.63 N047Y 0.45 A048F 164 2.51 A048G 0.83 A048H 165 1.99 A048I0.64 A048K 166 1.28 A048M 0.76 A048N 167 4.25 A048Q 1.05 A048R 0.66A048S 1.06 A048V 0.60 A048Y 0.81 T049I 0.42 T049K 0.85 T049R 168 1.41T049S 0.92 T049V 0.45 G050A 0.93 G050C 0.41 G050D 169 1.37 G050E 0.78G050H 0.74 G050L 0.43 G050M 171 0.47 G050Q 0.86 G050R 0.86 G050S 1701.24 G050V 0.3 G050Y 0.58 Q051N 0.60 Q051S 0.46 G052N 172 0.89 G052P0.43 G052Q 173 3.71 G052R 174 0.53 G052S 175 1.32 E375I 0.36 F380V 0.39G052T 176 0.49 T054A 0.43 T054F 0.56 T054N 0.48 T054Q 0.91 T054S 0.70T054V 0.66 V058C 177 0.55 V058G 0.54 V058H 183 1.09 V058I 0.57 V058K 1784.08 V058L 179 1.54 V058N 184 0.49 V058P 180 0.90 V058Q 181 4.54 V058R182 1.92 V058S 0.83 V058W 0.65 V058Y 185 1.07 D059Q 0.40 D059N 186 1.27R060K 0.69 L061I 0.42 L061M 0.73 L061V 0.59 Y063A 0.63 Y063H 1.07 Y063I1.03 Y063K 187 1.36 Y063L 188 1.33 Y063M 189 1.32 Y063N 0.96 Y063R 1901.40 Y063S 1.00 Y063T 1.07 Y063V 0.43 Y063W 191 1.53 P065R 0.57 Y066H0.47 Y066R 0.51 I067F 1.00 I067L 0.45 I067R 0.24 I067V 192 1.80 I067Y0.55 D068E 0.72 D068H 193 2.06 D068K 1.08 D068L 0.43 D068P 194 0.50D068Q 195 1.67 D068R 0.70 D068S 0.81 D068T 0.75 S069A 196 22.06 S069C197 1.97 S069E 198 1.48 S069F 199 8.75 S069G 200 6.06 S069I 201 3.12S069L 202 3.44 S069M 203 2.67 S069P 204 8.14 S069R 205 14.06 S069T 2060.58 S069W 207 2.18 S069Y 208 2.71 I070A 209 27.00 I070C 210 2.57 I070F211 5.69 I070G 212 6.22 I070H 213 9.09 I070K 214 14.64 I070L 215 3.05I070N 216 6.19 I070P 217 3.03 I070R 218 13.95 I070S 219 3.63 I070T 2205.43 I070V 221 6.34 I070Y 222 1.26 T071A 0.86 T071D 0.50 T071G 223 1.41T071H 0.93 T071L 1.09 T071M 0.89 T071N 224 1.21 T071Q 0.68 T071R 2252.17 T071S 226 1.54 G072A 0.45 G072D 0.60 S395W 0.4 G072E 0.69 G072H0.46 G072K 227 1.39 G072L 0.43 G072M 228 3.11 G072Q 229 2.33 G072R 0.65G072S 0.51 V073A 230 1.38 V073C 0.84 V073D 0.94 V073G 1.17 V073H 2311.54 V073K 232 1.42 V073L 233 1.59 V073M 0.68 V073Q 234 0.96 V073R 2350.72 V073S 0.86 K297R 0.34 S401Q 0.39 V073T 236 1.34 V073W 237 1.91T074A 238 2.28 T074C 239 2.18 T074E 240 1.38 T074F 241 1.43 T074G 2422.75 T074H 243 1.40 T074K 244 1.29 T074L 245 1.43 T074M 246 0.52 T074N247 2.12 T074P 248 2.45 T074R 249 2.22 T074S 250 1.80 T074V 251 2.27T074W 252 2.13 V075A 0.71 V075C 0.46 V075F 253 2.00 V075H 0.62 V075L 2545.22 V075M 255 1.16 V075N 0.81 V075Q 1.51 V075R 256 3.02 V075S 0.76V075T 257 4.34 V075Y 0.63 G077H 0.32 I079L 258 1.44 I079T 0.79 I079V1.01 Q081P 0.60 K082A 0.94 K082E 0.50 K082G 0.64 K082H 0.44 K082I 1.01K082L 259 0.87 K082M 0.58 K082N 260 0.96 K082Q 0.76 K082R 0.85 K082S0.62 K082T 0.56 K082Y 0.32 K082V 0.57 I083F 0.57 I083G 264 1.05 I083L0.93 I083N 0.82 I083Q 262 1.07 I083R 0.45 I083S 263 0.79 I083T 0.95I083V 261 0.99 S084D 0.98 S084E 265 0.52 S084F 266 0.72 S084G 267 8.68S084H 0.96 S084I 0.90 S084L 0.92 S084M 0.77 S084N 268 0.89 S084P 0.57S084Q 0.86 S084R 269 1.89 S084T 0.82 S084W 0.86 S084Y 0.30 S221I 0.35L085V 0.42 Q086A 270 2.70 Q086D 0.88 Q086E 1.18 Q086F 0.54 Q086G 1.02Q086H 271 1.70 Q086I 0.65 Q086K 272 0.97 Q086L 0.92 Q086M 1.06 Q086N 2731.28 Q086P 0.42 Q086R 0.93 Q086S 274 0.85 Q086T 275 0.58 Q086V 0.97Q086W 276 1.21 D087A 1.00 D087C 277 1.77 D087E 0.86 D087G 278 1.00 D087H0.72 D087I 0.53 D087L 279 0.55 D087M 280 0.58 D087P 0.31 D087Q 1.05D087R 281 1.28 D087S 282 0.99 D087T 283 1.70 A412H 0.39 D413H 0.31 V414K0.3 K415V 0.39 D087V 284 0.66 D087Y 285 2.72 L089C 286 1.46 L089R 0.34L089K 0.45 L089M 0.63 D090A 287 1.48 D090E 288 1.15 D090G 0.41 D090H 2891.24 D090I 1.10 D090K 290 1.36 D090L 1.15 D090N 291 1.18 D090Q 1.11D090R 292 1.49 D090S 1.15 D090T 1.02 D090W 0.81 K091A 0.89 K091Q 0.43K091R 0.67 A092C 293 1.97 A092H 0.22 A092L 294 1.29 A092M 0.86 A092T0.70 A092V 1.09 K093D 0.71 K093E 0.83 K093F 0.50 K093G 0.97 K093H 0.61K093I 295 3.25 R248H 0.4 K093L 296 1.53 K093M 0.70 K093N 0.71 K093Q 2970.84 K093R 298 1.52 K093S 299 1.25 K093T 300 3.93 K093V 0.24 K094A 0.64K094D 301 0.93 K094E 0.79 K094F 0.59 K094H 0.72 K094L 0.52 K094M 0.66K094N 0.99 K094Q 302 1.22 K094R 303 3.94 K094S 0.94 K094T 1.14 I096D0.69 I096L 0.46 I096V 0.68 T097A 304 1.25 T097C 305 0.53 T097D 306 1.31T097E 307 1.19 T097F 0.75 P257C 0.36 D426K 0.26 G427H 0.35 T097G 3084.84 T097I 0.85 T097L 309 1.22 T097N 1.10 T097P 0.62 T097Q 1.17 T097R0.95 T097S 310 1.21 T097W 0.53 T097Y 0.74 F098A 0.60 F098C 0.58 F098D0.47 F098E 0.44 F098H 1.06 F098I 0.52 F098L 0.58 F098M 0.87 F098Q 0.65P436C 0.39 F098R 0.72 F098S 0.56 F098V 0.46 F098W 0.81 Y099A 0.33 Y099R0.53 Y099S 0.43 V102A 0.83 V102C 0.69 V102E 0.90 V102G 0.67 V102H 0.88V102K 1.03 V102L 0.71 V102M 0.77 V102N 1.02 V102Q 1.03 V102R 0.94 V102S311 1.41 V102T 312 1.26 V102W 0.76 D103N 0.39 N104A 0.69 N104C 0.41N104G 0.48 N104K 0.88 N104M 0.61 N104R 313 1.25 N104S 1.03 N104T 0.71L105A 0.54 L105G 0.51 L105I 0.94 L105P 0.84 L105Q 0.90 L105R 0.65 L105S0.61 L105T 0.51 L105W 0.34 L105V 0.99 G106V 0.43 M107F 0.91 M107I 0.67M107L 314 1.32 A108G 0.47 I110V 0.51 E114A 315 1.44 E114G 0.73 E114H0.75 E114M 0.44 E114S 0.69 P117D 0.56 T118H 0.47 T118K 0.53 T118L 1.09T118M 0.53 T118N 0.67 T118Q 316 3.37 T118V 0.79 W119F 0.53 W119P 0.36W119Y 1.08 A120D 0.76 A120F 318 2.62 A120G 1.03 A120H 317 1.11 A120I 3191.33 A120L 1.25 A120N 0.81 A120P 0.42 A120R 0.82 A120S 320 1.21 A120T0.62 A120V 321 1.53 A120W 0.59 A120Y 322 1.95 N122M 0.56 K124L 0.34K124R 0.62 P125H 0.43 P125R 0.63 P125S 0.54 D127A 0.89 D127E 323 1.31D127G 0.97 D127H 324 2.33 D127L 0.84 D127M 0.4 D127N 325 1.69 D127Q 3261.21 D127R 327 0.51 D127S 0.77 D127T 1.11 D127V 0.56 D127W 0.44 V128A0.53 V128C 0.68 V128G 0.49 V128I 328 1.25 V128K 1.16 V128L 0.95 V128Q0.55 V128R 0.74 V128S 0.53 V128W 0.50 K130I 0.50 K130R 329 1.42 N131C0.60 N131E 0.44 N131F 0.63 N131G 330 2.47 N131H 0.80 N131I 331 1.40N131L 0.82 N131M 332 0.99 N131Q 333 1.24 N131R 334 2.81 N131S 0.76 N131T1.02 N131V 335 2.08 N131Y 0.85 R132A 0.68 R132C 0.58 R132E 0.70 R132F0.60 R132H 0.66 K279A 0.27 E285A 0.34 R132I 0.56 R132K 1.05 R132L 3370.76 R132N 336 1.28 R132Q 0.69 R132S 0.79 R132T 0.61 R132V 0.73 R132Y0.78 S133I 0.54 I134L 1.04 I134T 0.60 I134V 1.08 E135A 0.99 E135C 0.77E135D 338 2.68 E135F 0.73 E442L 0.4 E135G 339 2.79 E135H 0.79 E135K 1.15E135L 0.82 E135N 0.56 E135Q 1.59 E135R 340 2.08 E135S 1.13 E135W 0.63E135Y 0.50 L136A 0.73 L136C 0.56 L136D 0.47 L136F 0.96 L136H 1.00 L136I0.65 L136M 1.05 L136N 0.48 L136Q 0.61 L136R 0.74 L136S 0.80 L136T 0.72L136W 1.11 V137A 0.48 V137I 1.01 V137T 0.51 Q138A 0.69 Q138C 0.65 Q138H0.71 Q138I 0.54 Q138L 341 0.59 Q138M 0.68 Q138N 0.61 Q138R 0.53 Q138S0.48 Q138W 0.41 Q138Y 0.60 Q139A 0.92 Q139C 0.44 Q139D 0.48 Q139E 0.94Q139F 0.53 Q139G 0.65 Q139H 0.56 Q139K 0.73 Q139L 0.70 Q139M 0.95 Q139R0.79 Q139S 0.81 Q139T 342 1.31 Q139V 0.77 Q140A 0.96 Q140C 0.50 Q140D0.59 Q140F 0.66 Q140G 0.73 Q140H 0.84 Q140I 0.75 Q140K 343 0.93 Q140L0.51 Q140M 0.80 Q140R 0.85 Q140V 0.61 Q140W 0.59 Q140Y 0.41 N141A 1.12N141D 1.09 N141E 0.67 N141F 0.81 N141G 1.15 N141H 344 2.03 N002I 0.37G297A 0.57 N141L 0.61 N141M 0.48 N141Q 1.16 N141R 345 1.40 N141S 3460.72 N141T 0.45 N141V 0.50 N141W 347 0.83 N141Y 348 1.55 V142C 0.61V142D 349 0.71 V142E 0.87 V142G 350 0.98 V142H 1.11 V142I 0.81 V142K 3511.40 V142L 0.75 V142M 0.76 V142N 352 0.98 V142P 353 0.88 V142Q 354 1.04V142R 355 1.53 V142S 356 0.93 V142T 357 1.19 Q143E 0.77 Q143G 358 0.62Q143I 0.44 Q143K 359 1.30 I009Q 82 0.4 Q143L 0.56 Q143N 0.73 Q143V 0.57L144T 361 1.02 L144W 0.79 S145A 0.58 S145C 0.44 S145D 0.48 S145E 0.56S145G 0.94 S145H 0.56 S145L 0.44 S145M 0.56 S145N 0.58 S145P 1.04 S145R0.97 L146A 0.52 L146C 0.42 G305N 0.36 M310Y 0.38 L146E 0.50 L146G 0.62L146H 0.78 L146I 0.82 L146K 0.84 L146N 0.57 L146P 362 0.93 L146Q 0.84L146R 363 1.47 L146S 0.71 L146T 0.74 L146V 0.84 L146Y 0.80 S312K 0.38T147A 364 1.20 T147C 0.47 T147D 0.71 T147F 365 1.24 T147G 1.05 T147I0.85 T147L 366 1.30 T147M 0.79 T147P 1.09 T147Q 1.29 T147R 367 2.11T147S 368 1.27 T147V 369 2.04 T147W 0.97 T147Y 1.04 E148C 0.66 E148F0.42 E148G 1.05 E148H 370 1.24 E148I 0.73 E148K 371 1.63 E148L 0.85E148Q 372 1.44 E148R 0.97 E148S 1.15 E148T 0.82 E148V 0.99 E148W 0.43E148Y 0.95 A149C 1.15 A149G 0.52 A149K 0.51 A149L 0.88 A149M 0.88 A149Q1.15 A149R 1.02 A149S 1.08 A149T 373 1.24 A149V 374 1.34 T150A 375 1.21T150C 0.70 T150D 376 1.24 T150E 1.05 T150F 0.71 T150G 377 2.19 T150I0.52 T150L 0.70 T150N 378 0.91 T150P 0.88 T150R 0.90 T150S 379 0.92T150W 380 1.25 T150Y 381 1.36 E151A 382 1.27 E151C 1.00 E151G 1.06 E151H383 1.34 E151K 384 2.05 E151L 385 1.03 E151M 386 1.26 E151N 0.95 E151Q387 2.01 D320L 0.37 E151R 388 1.61 E151S 389 1.28 E151T 390 1.21 E151V391 1.38 E151W 392 1.31 E151Y 393 1.31 K152A 0.51 K152C 0.52 K152F 0.61K152I 0.65 K152M 0.75 K152R 394 1.85 K152T 395 1.20 K152V 0.82 K152Y0.67 A153I 0.93 A153L 0.51 K154R 0.86 K154T 0.83 K154V 0.46 Q155A 0.91Q155C 0.60 Q155D 397 1.49 Q155F 0.70 Q155G 398 1.61 Q155H 1.03 Q155K 3991.57 Q155L 0.86 Q155M 0.97 Q155R 400 1.27 Q155S 0.77 Q155T 0.76 Q155V0.73 Q155W 0.91 E156A 0.79 E156D 401 1.95 E156G 0.49 E156I 0.51 E156L0.43 E156M 0.87 E156Q 0.84 E156R 0.43 E156S 0.62 E156T 0.69 E156V 0.45E156W 0.49 F157W 0.61 E158A 0.56 E158F 0.51 E158H 0.54 I326S 0.95 I331E0.34 E158L 0.44 E158Q 402 1.25 E158S 403 0.95 K159A 0.64 K159D 0.52K159E 0.49 K159H 0.74 K159L 0.62 K159M 0.66 K159N 0.73 K159Q 0.92 K159R0.88 K159S 0.67 K159V 0.41 A160C 0.61 A160F 0.79 A160G 0.75 A160H 0.47A160I 0.43 A160K 0.91 A160L 0.67 M035Q 0.37 A160M 0.77 A160N 0.56 A160Q0.65 A160R 0.89 A160S 404 1.35 A160V 0.73 A160Y 1.07 G161A 0.99 G161C0.44 G161D 0.86 G161E 0.49 G161R 0.48 S036N 148 0.38 G161S 0.77 G161V0.42 K162A 0.50 K162D 0.77 K162E 405 0.51 V351C 0.35 W357K 0.36 K162G0.56 K162H 0.62 K162L 0.54 K162M 1.04 K162P 0.64 K162Q 0.58 K162R 0.52K162S 0.47 K162V 0.52 K162W 1.01 K162Y 0.72 D163A 406 1.52 D163E 4071.63 D163G 1.15 D163K 408 1.90 D163L 1.18 D163Q 409 1.40 D163R 410 1.80D163S 411 1.34 D163T 1.13 D163V 0.76 F164L 1.13 F164M 412 1.66 F164V 4131.23 S043N 0.34 F164W 0.72 L165A 0.48 L165D 414 5.79 L165F 415 1.23L165N 416 2.19 L165R 0.59 L165S 417 1.31 L165V 418 1.22 L165W 1.14 A371G0.38 L165Y 0.66 V166A 419 2.85 V166C 1.16 V166E 420 1.28 V166F 421 1.67V166G 1.11 V166H 422 1.74 V166L 423 4.38 V166Q 424 3.61 V166R 425 5.56V166T 426 4.26 V166W 427 1.26 V166Y 428 2.08 E167A 0.84 E167D 429 0.69E167G 0.60 E167H 0.89 E167K 0.91 E167M 0.87 E167N 0.83 E167P 0.58 E167R1.02 E167S 1.17 E167T 0.59 E167Y 0.55 T168H 0.46 I169L 430 2.08 I169R0.54 I169V 0.74 K170N 0.72 K170R 431 2.58 K170V 0.58 L171I 0.73 L171V0.64 G172A 432 1.20 G172C 1.03 K173N 0.44 K173R 433 0.82 L174A 1.20L174G 434 0.40 L174K 435 2.39 L174M 0.79 L174N 436 1.36 L174Q 0.99 L174R437 1.50 L174S 0.85 L174T 438 1.12 L174V 0.62 L174W 0.78 L174Y 1.06L175E 0.43 Q051A 0.34 L175H 0.57 L175T 439 1.43 L175V 0.94 L175Y 0.66R176K 0.67 N178G 0.85 N178K 440 0.85 N178M 0.88 K376L 0.37 F380T 0.39N178R 441 1.10 H179A 1.06 H179C 0.94 H179E 0.62 H179G 0.86 H179I 0.90H179K 442 1.39 H179L 0.73 H179M 0.63 H179N 0.96 H179P 0.44 H179R 0.96H179S 0.51 H179T 0.43 H179V 0.42 L180F 0.59 L180G 0.62 L180K 0.44 L180M0.64 W181M 0.88 L061F 0.3 W181Q 0.88 G182L 0.90 Y183L 0.70 F186Y 0.59H192S 0.49 H192T 0.50 H193G 0.68 H193Q 443 0.82 H193S 0.42 H193Y 0.58K195A 0.51 K195G 0.45 K195H 0.45 K195I 0.50 K195L 0.45 K195N 445 0.74K195Q 0.71 K195R 0.85 K195S 0.42 K195T 444 0.58 K195W 0.49 K196E 4460.43 D068G 0.37 K196G 0.41 K196L 0.65 K196R 447 0.58 K196S 0.68 K196T1.18 K196W 0.55 P197A 0.81 P197D 0.58 P197E 0.52 P197F 0.48 P197G 0.75P197H 0.62 P197K 0.99 P197L 0.56 P197M 1.03 P197Q 0.69 P197R 0.58 P197S0.70 P197T 0.41 G198A 0.80 G198D 448 1.99 G198E 0.49 G198H 0.84 G198L0.48 G198N 0.80 G198Q 0.55 G198R 0.58 G198S 0.76 G198T 0.41 G198Y 0.81N200D 0.46 S202M 0.40 F204P 449 0.63 N205A 450 1.30 N205D 0.85 N205E 4511.94 N205F 0.52 N205G 0.79 N205K 0.76 N205M 0.58 N205P 0.75 N205R 0.54N205S 0.80 N205T 453 0.85 N205V 0.49 N205W 0.41 V206H 0.50 V206I 4540.94 V206K 455 1.75 V206L 456 1.57 S395T 0.39 V206M 0.43 V206R 457 1.30V206S 0.72 G072Y 0.35 V206T 0.59 I208A 0.62 I208C 0.48 I208K 0.91 I208L0.84 I208M 0.88 I208Q 0.77 I208R 1.14 I208S 0.62 I208T 1.01 I208V 1.07K209A 0.53 K209E 0.46 K209G 0.44 K209N 0.50 F398L 0.35 S404T 0.37 K209R458 0.68 K209S 0.50 K209T 0.50 D212N 459 1.52 D212S 460 0.93 D212T 0.76D213A 461 0.85 D213E 0.79 D213G 0.81 D213H 0.75 D213K 0.82 D213L 0.56D213M 462 1.56 D213N 463 1.53 D213Q 1.04 D213R 0.92 D213V 0.47 D213W0.49 D213Y 0.49 L214Q 0.57 S215A 0.74 S215D 0.62 S215E 0.74 S215G 0.88S215H 464 0.91 S215K 0.99 S215L 0.60 S215M 465 1.77 S215Q 0.79 G077K0.32 S215R 0.71 S215T 0.80 S215V 0.69 S215W 0.52 W216Y 0.48 L217M 0.51W218F 0.57 N219A 466 1.29 N219C 0.43 N219D 0.75 N219E 0.95 N219H 0.97N219I 467 0.60 N219K 468 1.45 N219L 0.72 N219M 1.02 I083H 0.4 N219R 1.10N219S 469 2.48 N219T 0.82 N219W 0.48 E220A 0.75 E220H 470 1.40 E220I 4711.34 E220L 472 1.45 E220S 0.62 E220T 0.91 E220V 473 1.35 S221A 0.72S221C 0.59 S221M 0.46 S221Q 474 1.37 S221T 0.94 S221V 1.04 T222D 0.43T222F 0.43 T222G 475 0.49 T222K 0.75 T222L 0.64 T222N 0.80 T222R 0.75E220D 0.39 T222I 0.4 T222S 0.63 T222V 0.79 L224I 0.61 L230I 0.87 N231T1.10 T232F 476 0.73 T232S 0.76 Q233A 0.71 Q233F 0.53 Q233G 477 0.46Q233K 478 1.69 Q233L 0.69 Q233R 479 1.50 Q233Y 0.50 Q234M 480 1.65 S235A481 0.47 S235E 1.00 S235G 0.95 S235H 0.44 S235K 0.53 S235T 0.66 P236A1.07 P236G 1.09 P236H 0.46 P236K 0.71 P236R 482 3.09 Q234L 0.40 P236S0.91 V237A 0.90 V237E 484 1.93 V237F 0.41 A412Q 751 0.35 A413Q 0.38V414L 0.36 D418G 0.45 V237H 485 0.75 V237L 1.12 V237N 0.67 L089W 0.26V237Q 486 1.46 V237R 0.71 V237S 1.03 V237T 487 1.01 V237W 0.52 A238D0.75 A238E 488 0.59 A238H 489 0.60 A238K 0.60 A238Q 1.02 A238R 0.49A238S 490 2.62 A238T 0.44 T240K 1.13 T240A 491 0.48 T240M 0.48 T240P0.56 T240Q 492 0.75 T240R 0.91 A239N 0.32 T240S 0.74 T240V 0.77 Y242F1.08 N245H 0.50 V247I 493 2.01 V247L 0.83 V247M 0.52 R248A 494 0.43R248W 0.52 R248Y 0.67 I251Y 0.37 I251L 0.58 I251M 0.43 V253I 0.76 K255A0.40 K255N 0.52 K255Q 0.91 K255R 0.71 K093P 0.38 K255S 0.43 I256A 0.42I256H 0.51 I256L 0.64 I256V 0.51 P257A 0.82 P257G 496 0.51 P257I 1.07P257K 0.92 P257L 0.69 P257M 0.90 P257N 0.69 P257Q 0.61 P257R 498 1.38P257T 497 2.04 P257V 0.88 D258H 0.84 D258N 499 1.44 D258R 0.45 D258S 5001.44 D258G 0.39 D426S 0.36 G427I 0.54 A259E 0.85 A259G 0.68 A259I 0.46A259K 0.76 A259L 0.53 A259N 0.49 A259P 501 1.54 A259Q 0.70 A259R 0.72A259S 0.63 A259T 0.51 A259V 0.41 A259W 0.55 A259Y 0.51 K260A 0.66 K260D0.41 K260E 0.58 K260H 0.87 K260L 0.60 E249V 495 K260M 502 0.85 K260Q0.58 K260R 0.83 K260S 0.66 K260G 0.37 K260Y 503 1.73 S261A 504 0.74S261F 0.73 S261K 505 2.54 S261M 0.56 S261N 506 1.98 S261Q 0.76 S261R1.19 S261T 0.66 S261V 0.48 S261W 0.44 L263A 0.76 L263K 507 2.73 L263M0.89 L263R 508 1.63 L263T 0.49 N104I 0.35 L263V 0.75 P264A 0.43 P264H0.60 V265I 0.58 F266Y 0.58 A267M 0.45 A267T 509 1.34 T269A 510 1.63T269C 0.75 T269D 0.76 T269S 1.01 R270M 0.46 R270N 0.52 R270S 0.69 I271F0.72 I271G 1.29 L105C 0.33 I271L 511 10.62 V272E 0.39 I271M 512 3.24I271S 0.42 I271V 1.05 V272D 513 1.36 V272R 0.74 V272S 0.96 V272T 5141.61 F273H 515 1.41 F273T 0.48 F273Y 516 0.90 T274A 0.51 1274F 517 1.28T274S 0.62 Q276C 0.88 Q276D 518 1.69 Q276E 1.05 Q276H 519 1.20 Q276I0.51 Q276L 0.48 W119Q 0.72 Q276M 520 1.14 Q276R 521 1.30 Q276S 522 1.63Q276Y 523 1.94 V277A 524 0.65 V277C 0.41 V277D 0.79 V277E 525 1.02 V277G1.18 V277H 526 1.09 V277K 527 1.51 V277M 528 0.94 V277N 529 1.15 V277Q530 0.82 V277R 531 1.63 V277S 532 0.83 K124H 0.35 V277T 533 1.94 V277Y0.66 L278A 1.13 L278E 534 1.03 L278F 535 1.26 L278G 536 1.33 L278H 5374.50 L278I 0.93 L278K 538 1.75 D275V 0.4 L278N 539 1.74 L278R 540 5.87L278S 541 1.67 L278T 542 1.66 L278V 0.44 L278Y 543 1.51 K279H 544 0.44K279Q 0.84 K279R 1.10 K279T 0.86 F280G 0.47 F280Q 0.43 S282D 0.41 S282G0.54 S282M 545 2.64 S282Q 0.41 Q283E 0.63 Q283P 1.18 Q283R 0.59 Q283S546 1.73 Q283T 0.65 D284A 0.58 D284E 1.21 D284G 0.60 D284H 0.51 D284L0.50 D284M 0.56 D284N 0.40 D284Q 0.95 D284S 0.99 E285F 0.47 E285G 0.52E285H 547 1.30 E285M 0.43 E285N 0.40 E285Q 0.59 E285Y 0.99 L286S 0.46D284T 0.39 L286R 0.53 V287I 0.51 V287T 548 0.50 Y288L 0.79 Y288W 0.49T289K 0.75 T289S 549 0.48 F290I 0.41 F290M 1.03 G291Q 0.80 G291R 0.45G291S 550 0.41 G291V 551 1.63 E292A 0.66 E292C 552 0.71 E292F 553 0.90E292G 0.41 E292H 554 1.26 E442W 0.38 E292K 555 1.27 E292N 0.99 E292P1.05 E292R 556 0.42 E292V 557 1.28 E292W 0.83 T293A 558 1.90 T293C 5591.67 T293D 560 1.46 V137C 0.37 V137S 0.36 V137L 0.21 Q143C 0.28 L144R360 0.26 K152W 396 0.37 A153S 0.34 K154I 0.38 E156C 0.35 E158G 0.37K159G 0.38 A160W 0.39 G161V 0.42 D163W 0.38 D163F 0.39 L165C 0.27 V166N0.47 E167F 0.31 K170A 0.40 K170Q 0.40 K173Q 0.32 L174H 0.38 R176L 0.40P177V 0.36 L180I 0.38 W181K 0.29 Y183E 0.32 Y184W 0.39 H193R 0.33 H193F0.38 K195V 0.36 K196N 0.39 K196Y 0.39 P197W 0.39 G198W 0.29 N200T 0.37F204W 0.39 N205L 452 0.39 N205Y 0.4 V206Q 0.33 K209F 0.4 K209L 0.38N211L 0.41 N211W 0.51 W218M 0.38 W218V 0.28 T293F 561 1.94 T293G 1.00T293K 562 1.35 T293L 1.00 T293M 563 2.29 T293P 564 1.64 T293Q 565 1.83T293S 0.89 T293V 566 2.15 T293Y 567 1.49 V294M 0.41 A298G 568 0.43 A298I0.41 G300R 0.42 I301A 0.88 I301V 0.88 V287N 0.35 V302W 0.46 V302I 0.45I303V 0.47 W304G 1.13 W304I 1.17 G305D 1.00 G305E 569 1.62 T306D 0.76T306E 0.52 T306S 1.02 L307K 0.43 L307N 0.76 L307Q 0.61 L307S 0.86 L307T1.08 L307V 0.48 L307W 0.64 L307Y 0.60 S308D 571 0.92 S308G 572 1.73S308H 1.15 S308K 573 1.33 S308N 574 2.33 S308P 0.65 S308R 575 1.34 S308T0.72 I309D 0.72 I309E 576 1.99 I309G 577 1.44 I303D 0.34 I309H 578 1.30I309K 0.98 I309L 579 1.72 I309M 580 1.47 I309N 581 3.11 I309Q 582 1.64I309R 583 2.27 I309S 584 1.16 I309T 585 2.09 I309V 586 0.60 I309W 0.88M310A 587 1.50 M310G 588 2.73 M310Q 589 0.59 M310R 0.50 M310S 590 1.61M310V 0.70 R311G 0.53 L307G 570 0.32 R311G 0.54 R311H 0.48 R311K 0.72R311Q 0.43 R311S 0.84 R311T 0.52 S312G 0.49 S312N 1.26 S312T 0.75 M313A591 1.34 M313E 0.63 M313G 592 0.56 M313H 593 1.23 M313K 594 2.85 S312L0.38 M313L 1.05 M313P 595 1.11 M313R 596 2.30 M313S 0.88 M313T 597 0.67M313V 0.99 M313Y 598 1.12 K314A 0.82 K314D 0.53 K314H 1.10 K314I 0.54K314N 0.57 K314Q 0.62 K314R 0.95 K314S 599 0.61 K314T 0.61 K314Y 6000.45 S315A 601 0.85 S315E 0.41 S315G 0.72 S315H 602 2.04 S315K 0.62S315L 0.42 S315M 0.63 S315R 1.04 S315T 0.97 S315Y 603 0.50 C316D 0.41L317A 604 1.27 L317D 0.61 L317H 1.05 L317I 605 1.76 L317K 606 5.11 L317M1.20 L317N 607 0.73 L317Q 608 1.67 L317R 609 2.41 L317S 610 1.03 L317T611 0.93 L317W 612 0.84 L318D 614 0.46 L318F 0.51 L318G 0.49 L318H 6150.45 L318I 0.70 L318K 616 1.36 L318M 613 1.68 L318N 0.52 L318Q 0.71L318R 617 1.34 L318S 0.71 L318T 0.63 D320E 0.78 D320G 0.83 D320H 6181.75 D320I 1.00 D320K 619 6.42 D320M 0.79 D320N 0.52 D320R 620 3.19D320S 1.19 D320W 0.40 D320V 0.35 D320Y 0.86 N321A 1.01 N321D 1.25 N321H0.92 N321K 1.29 N321R 621 1.23 N321S 622 1.26 N321T 0.64 N321Y 0.40M323F 0.64 M323I 0.55 M323L 0.55 E324A 0.59 E324D 1.15 E324H 0.79 E324M0.50 E324N 623 1.01 E324R 624 2.28 E324S 0.62 T325A 625 1.87 T325D 6261.78 T325E 627 4.03 T325G 628 4.21 T325H 629 3.45 T325K 630 4.37 T325M631 2.11 T325N 632 4.64 T325Q 633 5.08 T325S 634 3.19 T325V 635 1.24T325W 0.62 I326K 0.95 I326L 636 1.50 I326V 637 6.29 I326Y 0.77 L327M0.52 N328A 0.67 N328C 638 1.25 N328G 639 0.56 N328H 0.88 N328I 642 1.85N328K 640 2.12 N328L 641 2.01 N328Q 1.13 N328R 0.68 N328S 643 2.22 N328T0.59 N328V 1.16 N328Y 644 1.66 I331V 0.94 N328W 0.33 V334T 0.39 V334P0.46 T335S 645 0.47 A338Q 0.63 K339M 0.61 S342A 0.68 Q343T 0.49 Q343V0.51 Q347A 646 0.78 Q347E 0.78 Q347G 647 2.68 Q347M 0.61 Q347R 0.55Q347S 648 2.38 E348D 0.67 E348G 0.55 E348S 0.44 Q349A 0.47 Q349E 0.83Q349K 0.93 Q349M 649 0.70 Q349N 0.44 M035V 146 0.37 Q349R 650 0.73 Q349T0.49 V351A 1.14 V351S 651 0.92 I353T 0.42 I353V 652 1.61 N356A 0.41N356D 0.79 N356H 653 0.82 N356S 654 0.46 W357A 0.80 W357C 0.67 L037W0.36 W357S 0.41 W357T 0.62 N358C 0.66 N358G 0.41 N358T 0.58 V351I 0.36N358L 0.38 S359D 0.45 S359E 655 1.05 S359H 656 0.44 S359K 0.66 S359M0.63 S359T 657 2.11 S359V 0.65 S360T 0.50 P367A 658 0.55 P367C 0.83P367G 659 0.47 P367K 660 0.57 P367R 0.46 P367S 661 0.52 D368A 662 1.34D368E 663 1.28 D368G 0.49 D368H 0.96 D368K 664 1.31 D368L 665 0.64 D368M666 0.78 D368R 667 1.31 D368S 0.93 D368T 668 0.80 D361H 0.37 D368V 0.41N369H 669 1.33 N369R 670 0.55 N369S 0.54 A371E 1.05 A371F 671 0.52 A371H672 1.20 A371I 0.50 A371K 673 1.76 L374W 0.34 A371L 674 0.57 A371M 0.57A371R 675 1.51 A371S 676 1.45 A371V 0.94 Q373A 0.65 Q373E 0.81 Q373F0.62 Q373K 0.73 Q373L 0.84 Q373M 677 1.43 Q373R 0.68 Q373S 0.87 Q373V1.05 L374A 0.60 L374H 678 1.42 L374I 0.80 L374M 1.11 L374N 0.43 L374P679 0.43 L374R 0.83 L374S 0.58 L374T 0.47 L374V 0.56 L374Y 0.66 E375A680 0.42 E375G 681 0.90 E375K 682 1.49 E375L 0.46 E375M 0.54 E375N 0.81E375R 683 0.43 E375S 0.77 E375T 1.17 K376A 0.95 K376D 684 0.78 K376E 6850.88 K376M 0.46 K376Q 686 0.69 K376R 687 0.67 K376S 0.80 K376T 688 0.53K376V 689 0.58 K376Y 690 0.42 G377D 691 1.35 G377E 692 0.59 G377H 6931.49 G377K 694 1.50 G377P 695 2.30 G377R 696 1.28 G377S 697 1.80 Q051R0.36 G377T 698 3.83 G378K 1.22 G378N 0.64 G378R 1.03 K379G 0.52 K379H0.57 K379R 0.74 K379S 0.46 K379T 0.4 M035Q 145 0.37 F380I 0.56 F380L0.67 F380P 0.47 F380W 699 2.15 F380Y 700 1.50 T381H 0.48 T381K 1.06T381N 0.51 T381Q 0.84 T381R 0.87 T381S 701 0.87 T381V 0.89 R383A 0.51R383E 0.51 R383H 0.71 R383I 702 0.71 R383K 703 1.30 R383L 704 1.31 R383M0.61 R383N 0.77 T381E 0.35 R383S 705 0.87 R383T 0.98 R383V 1.05 K385A706 1.12 K385G 0.62 K385H 0.50 K385N 0.41 K385Q 707 0.73 K385R 0.94K385S 1.05 K385T 0.46 K385V 708 0.43 T387S 0.93 L388F 0.92 L388H 0.47L388I 0.98 L388M 0.79 L388R 0.60 L388T 0.51 L388V 0.78 L388W 0.77 L388Y1.18 E392W 0.31 E389A 709 1.14 E389G 710 0.91 E389H 1.17 E389K 712 1.91E389L 711 0.65 E389M 0.60 E389P 0.75 E389Q 713 0.69 E389R 0.94 E389S 7141.08 E389T 0.70 E389Y 0.77 L391C 0.90 E392A 715 0.58 E392F 716 0.54E392G 1.00 E392K 0.66 E392L 0.80 E392M 717 1.54 E392Q 718 1.01 E392R 7190.66 E392S 0.52 E392T 0.72 E392V 720 1.27 E392Y 0.92 Q393A 1.26 Q393D0.45 Q393F 721 1.23 Q393H 1.05 Q393K 0.80 Q393L 0.91 Q393M 722 0.80Q393N 0.72 Q393R 0.74 Q393S 1.15 Q393T 0.41 F394L 0.56 F394W 0.41 S395A723 1.10 S395G 0.77 S395H 724 0.56 S395K 0.96 S395R 725 1.98 E396A 7260.52 E396D 0.64 E396H 727 0.47 E396Q 728 0.73 E396R 0.61 E396S 729 0.61E396T 0.89 E396L 0.39 Y399A 1.01 Y399C 0.46 Y399E 1.49 S407L 0.4 Y399K730 1.94 Y399M 731 2.70 Y399N 0.52 Y399Q 1.18 Y399R 1.20 Y399S 1.01Y399T 732 2.40 Y399V 733 1.44 Y399W 734 1.92 S401A 735 0.82 S401E 7360.46 S401N 0.42 Y403F 0.62 S404A 737 0.63 S404P 0.64 S401G 0.38 T405F0.36 T405A 0.56 T405G 738 2.32 T405K 0.74 T405M 0.48 T405P 0.64 T405Q0.75 T405R 0.60 T405S 0.94 T405W 0.73 T405Y 0.44 L406A 0.70 L406C 0.98L406E 0.73 L406F 739 1.42 L406G 1.00 L406I 0.61 L406N 740 0.76 L406Q0.93 L406S 0.47 L406T 0.83 L406V 0.87 L406Y 0.74 S407A 741 1.16 S407D742 1.52 S407E 743 1.38 S407F 744 1.42 S407G 0.75 S407H 745 1.34 S407M0.74 K411H 0.33 S407N 0.72 S407P 747 0.94 S407Q 746 1.71 S407R 1.04S407V 0.56 S407W 0.41 K409A 748 2.18 K409D 0.65 K409E 0.62 K409G 0.50K409H 0.64 K409I 0.51 K409P 0.48 K409Q 749 3.33 K409R 0.84 K409S 0.72I208K 0.30 K409T 0.63 K409V 0.48 A412Y 0.66 E410D 0.47 E410K 0.70 E410M0.42 E410N 0.67 E410P 0.73 E410Q 0.85 E410R 0.61 E410S 0.81 E410T 7501.54 E410V 0.65 E410Y 0.62 K411A 0.48 K411N 1.02 K411P 0.42 K411R 0.97K411S 1.21 K411T 0.63 K411V 0.99 A412D 0.74 A412G 0.80 A412I 0.81 E220M0.36 P226W 0.51 A412L 0.65 A412N 0.86 A412P 0.77 A412R 752 0.66 A412S0.86 A412V 753 0.53 A412W 0.54 D413E 0.52 D413K 0.42 D413N 0.94 D413R0.50 D413T 0.41 V414I 1.12 V414M 0.53 K415G 0.40 K415S 0.42 K415W 0.42D416F 0.41 D416G 0.67 D416H 0.57 D416I 0.63 D416K 0.76 D416L 754 0.75D416N 0.73 D416Q 0.83 D416R 0.46 V237C 483 0.35 D416T 0.85 D416V 0.59D416Y 0.40 T417I 1.22 D413A 0.38 D413S 0.39 K415Y 0.39 D418A 0.92 D418E755 1.31 D418F 0.81 L089P 0.38 D418G 0.45 D418I 0.99 D418L 756 1.28D418M 1.09 D418N 0.91 D418P 757 2.11 D418Q 1.05 D418R 758 1.18 D418S0.78 D418V 759 1.43 D418Y 0.97 A419E 0.45 A419F 760 2.17 A419G 0.42A419H 761 1.21 A419I 762 1.64 A419K 763 1.88 A419L 0.56 A419N 0.53 V421I0.39 A419R 764 1.81 A419S 765 2.65 A419W 0.69 A419Y 766 1.44 V420I 1.04V420P 0.48 D421A 767 1.28 D421E 0.81 D421G 0.62 D421H 768 1.98 K255G0.39 D421K 769 2.42 D421L 0.73 D421M 0.94 D421N 770 1.89 D421Q 771 1.54D421R 772 2.21 D421S 773 2.12 K094C 0.33 D421T 0.80 D421Y 0.66 V422I0.42 V422T 0.49 A425G 774 1.20 A425I 0.44 A425K 775 1.75 A425M 0.70A425N 0.46 A425R 0.49 A425S 0.47 D426E 0.62 D426G 0.85 D426N 0.61 D426P1.03 D426Q 0.42 D426Y 0.43 G427K 0.52 G427S 0.42 V428L 778 1.25 A425Y0.39 G427T 777 0.35 G427Q 776 0.39 V428M 0.42 V428P 0.82 V428T 0.62D431A 779 2.42 D431E 781 1.27 D431G 780 0.55 D431H 782 3.13 D431I 1.05D431K 783 1.83 D431L 784 0.62 D431N 785 1.30 D431Q 786 2.16 D431R 7872.20 D431S 788 1.91 D431V 789 1.52 D431W 0.56 D431Y 0.85 A432E 0.60A432G 0.52 A432H 0.34 A432N 0.51 A432S 0.61 A432V 0.56 F433A 790 0.97R270T 0.40 F433C 0.69 F433D 0.95 F433E 0.82 F433G 0.54 F433H 791 0.83F433I 792 1.06 F433K 793 1.36 F433L 794 1.87 F433P 0.95 F433R 795 1.63F433S 0.86 F433T 796 1.86 F433V 797 1.63 F433W 798 1.28 L434F 0.41 L434G0.47 L263H 0.36 L434I 0.89 L434M 0.60 L434V 0.46 K435A 1.08 K435C 0.53K435E 0.78 K435G 0.64 K435H 1.05 K435R 1.01 K435S 1.03 K435T 0.73 K435V0.44 K435Y 0.50 P436D 1.19 P436E 0.74 P436G 1.19 L105H 0.36 P436H 0.72V272M 0.31 P436I 0.84 P436K 799 2.05 P436L 0.63 P436M 0.61 P436Q 0.86P436R 1.00 P436S 0.92 P436T 0.59 P436W 0.43 P436Y 0.49 P437A 0.56 P437D0.62 P437G 0.50 P437H 1.11 P437I 800 2.46 P437K 0.83 P437L 0.51 P437M801 2.55 P437Q 0.96 D275L 0.24 P437R 0.85 P437S 0.57 P437Y 0.42 M438A802 0.75 M438C 0.63 M438D 803 0.87 M438E 804 0.72 M438G 0.83 M438L 8050.86 M438N 806 1.08 M438P 0.81 M438Q 0.85 M438R 0.99 M438S 0.83 M438T807 3.99 M438V 0.85 P125A 0.36 M438W 0.57 E439A 808 1.20 E439C 809 0.58E439F 1.00 E439G 1.22 E439H 0.74 E439K 810 1.20 E439L 0.88 E439P 8111.16 Q276G 0.36 E439Q 812 1.32 E439S 1.02 E439T 813 1.15 E439V 814 1.57E439W 0.62 T440A 1.22 T440D 815 1.03 T440E 1.00 T440F 0.85 T440G 0.86T440H 816 3.00 T440I 1.04 T440L 0.97 T440M 817 1.08 T440P 818 0.88 T440R819 1.77 T440S 820 1.17 T440V 1.02 T440Y 1.11 E441A 821 1.47 E441D 0.67E441F 822 3.91 E441G 0.87 E441H 0.65 E441K 0.80 E441L 0.82 E441N 0.82E441Q 0.81 E441S 0.79 E441T 0.66 E441V 0.54 E441Y 0.51 E442C 823 1.38E442G 824 0.51 E442H 0.76 E442K 0.73 E442P 0.91 E442Q 0.74 D284Y 0.37L286W 0.38 E442R 825 3.94 E442T 0.61 E442V 0.65 E442Y 0.60 P443A 8261.63 P443E 827 1.07 P443F 828 0.70 P443G 829 1.12 P443H 1.08 P443L 1.19P443M 830 1.99 P443N 831 1.25 P443Q 0.96 P443R 1.04 P443S 0.99 P443T0.87 P443W 0.64 Q444M 0.37 Q444D 0.97 Q444E 832 1.19 Q444F 0.66 Q444G0.93 Q444H 833 0.97 Q444I 0.58 Q444K 1.03 Q444N 1.01 Q444R 0.85 Q444V834 1.12 Q444W 0.64 Q444Y 0.67 I445A 0.97 I445G 0.98 I445H 835 1.35I445L 1.06 I445M 836 1.57 I445N 837 1.24 I445P 838 1.67 I445Q 839 1.26I445R 1.08 I445S 840 1.21 I445T 841 1.38 I445V 842 1.25 I445W 843 0.69I445Y 0.53 F446A 844 1.58 F446C 0.75 F446D 1.18 F446E 1.10 F446G 1.12F446H 1.28 F446I 1.06 F446K 0.94 F446L 0.93 F446M 845 1.31 F446Q 0.72F446R 0.89 F446T 0.89 F446V 0.91 F446W 846 1.40 Y447D 847 3.25 Y447E 8481.36 Y447F 1.41 Y447G 849 0.92 Y447I 850 1.36 Y447L 1.09 Y447M 0.90Y447N 851 1.58 Y447P 852 1.46 Y447Q 853 2.37 Y447R 1.12 Y447T 854 1.90Y447V 855 1.38 Y447W 1.07

2. Inactive Mutants

The other mutants that exhibited less than 20% hyaluronidase activity ofwildtype PH20, in at least one of the duplicates, were rescreened toconfirm that the dead mutants are inactive. To confirm the inactivemutants, the hyaluronidase activity assay described in Example 3 wasmodified to incorporate an overnight 37° C. substrate-sample incubationstep prior to measurement of enzymatic activity. The modified assay isintended to detect PH20 activities below 0.2 U/mL.

The preparation of the bHA coated plates and blocking of the platesprior to addition of the transfected variant supernatants or wildtypePH20 was the same as described in Example 3. The assay was modified asfollows. First, transfected variant supernatants or wildypte PH20 notcontaining a mutation generated as described in Example 2 were dilutedin duplicate 1:25 in assay diluent. For the standard curve, 1:3 serialdilutions of rHuPH20 (generated as described in Example 1) were made inassay diluent in duplicate starting from 0.1 U/mL down to 0.00014 U/mL.A blank well also was included. Then, 100 μl of the diluted samples orstandard were added to pre-designated wells of the bHA-coated andblocked plate and allowed to incubate at 37° C. overnight. After theincubation, the plates were washed and binding to bHA detected asdescribed above in Example 3. Optical density was measured at 450 nmwithin 30 minutes of adding the stop solution.

The identified reconfirmed inactive mutants are set forth in Table 10.The Table sets forth the amino acid replacement compared to the sequenceof amino acids of PH20 set forth in SEQ ID NO:3.

TABLE 10 Inactive Mutants N002H R060V R121W C189P P236I V287N L336WG377V N002K R060Y R121Y C189R P236L V287P L336Y G378D N002W L061A N122AC189S P236N V287Q A337C G378E N002Y L061E N122C C189T P236Q V287R A337FG378F F003A L061F N122E C189V P236T V287S A337G G378I F003G L061G N122FC189W P236Y Y288D A337I G378L F003K L061H N122I C189Y A238F Y288E A337KG378M F003P L061N N122K Y190C A238G Y288F A337L G378Q F003T L061P N122QY190E A238L Y288G A337M G378T F003V L061Q N122R Y190F A238P Y288H A337RG378W R004D L061R N122S Y190G A238V Y288I A337T G378Y R004E L061T N122TY190H A238W Y288K A337W K379A R004F L061W N122V Y190K A238Y Y288P A338CK379C R004G L061Y W123A Y190L A239C Y288R A338D K379E R004L G062A W123CY190N A239F Y288T A338E K379F R004P G062C W123D Y190Q A239G T289A A338FK379I R004W G062D W123E Y190R A239H T289C A338G K379L R004Y G062F W123HY190S A239I T289E A338H K379M A005D G062I W123L Y190T A239L T289G A338IK379W A005G G062K W123M Y190V A239P T289H A338K F380C A005I G062L W123PY190W A239R T289L A338L F380D A005L G062M W123Q N191A A239S T289P A338PF380E A005M G062P W123R N191E A239T T289Q A338R F380G A005N G062Q W123SN191F A239V T289R A338T F380Q A005P G062R W123T N191G A239W T289S A338VF380R A005Q G062S W123V N191K A239Y T289Y K339D F380S A005R G062T W123YN191L T240E F290D K339E T381G A005T G062V K124C N191M T240F F290Q K339FT381L A005V G062Y K124D N191P T240G F290Y K339G T381P A005W Y063C K124EN191Q T240N G291A K339H T381W A005Y Y063G K124F N191R T240W G291C K339LT381Y P006E Y063P K124N N191S T240Y G291D K339N V382E P006F Y064A P125CN191T L241A G291E K339P V382G P006T Y064C P125D N191V L241C G291F K339SV382H P006V Y064D P125G N191W L241D G291M K339T V382K P006Y Y064E P125LN191Y L241E G291N K339V V382L P007C Y064F P125N H192C L241G G291T K339WV382M P007D Y064G P125W H192F L241I G291W K339Y V382N P007F Y064H K126FH192G L241P G291Y M340A V382P P007G Y064I K126H H192K L241R E292I M340CV382Q P007H Y064K K126I H192L L241S E292L M340D V382R P007I Y064L K126LH192M L241T E292T M340E V382S P007K Y064P K126N H192N L241V T293E M340FV382T P007L Y064Q K126P H192P L241W T293N M340G V382W P007Q Y064R K126YH192Q Y242A V294A M340H V382Y P007R Y064S D127K H192R Y242C V294E M340KR383G P007S Y064T V128E H192V Y242D V294G M340P R383P P007T Y064V V128PH192W Y242G V294H M340R G384C P007W Y064W Y129A H192Y Y242I V294K M340SG384F P007Y P065A Y129C H193A Y242L V294L M340T G384M V008D P065C Y129DH193D Y242M V294N M340V G384Q V008E P065D Y129E H193K Y242P V294P M340WG384S V008G P065G Y129G H193L Y242R V294Q C341A G384T V008H P065H Y129HH193M Y242S V294R C341E K385C V008N P065I Y129L H193P Y242T V294S C341GK385L V008R P065K Y129P H193V Y242V V294T C341H K385M V008S P065N Y129QY194A Y242W V294W C341K K385P V008W P065R Y129S Y194C V243C A295C C341LK385W I009C P065S Y129T Y194I V243D A295G C341M K385Y I009D P065T Y129VY194L V243F A295H C341N P386A I009E P065V Y129W Y194P V243G A295I C341QP386C I009G P065W K130C Y194S V243H A295L C341R P386F I009N P065Y K130DY194T V243L A295N C341S P386G I009P Y066A K130G Y194V V243M A295P C341TP386H P010F Y066C K130H K195S V243P A295T C341V P386I P010I Y066D K130LP197C V243Q A295V C341Y P386L P010L Y066E K130N G198V V243R A295Y S342DP386M P010M Y066G K130S G198W V243S L296C S342E P386N P010Y Y066I K130TY199E V243W L296F S342F P386Q N011A Y066K K130W Y199G V243Y L296G S342HP386R N011C Y066L K130Y Y199H R244A L296I S342K P386S N011F Y066N N131PY199I R244D L296K S342L P386T N011I Y066P R132P Y199K R244G L296M S342MP386V N011L Y066S S133D Y199L R244I L296Q S342P P386Y N011P Y066T S133EY199P R244V L296R S342Q T387C N011T Y066V S133F Y199R R244Y L296S S342RT387E N011W I067D S133G Y199S N245A L296T S342T T387F N011Y I067E S133HY199W N245C L296V S342Y T387G V012G I067G S133L N200A N245F L296W Q343CT387H V012H I067P S133M N200F N245L L296Y Q343D T387I V012W I067R S133NN200G N245P G297C Q343F T387L P013E I067T S133P N200H N245Q G297E Q343IT387M P013G I067W S133R N200K N245R G297H Q343P T387N P013I D068A S133TN200L N245S G297L Q343W T387V P013L D068C S133V N200M N245T G297N V344FT387W P013M D068G S133W N200P N245V G297P V344G T387Y P013V D068I I134AN200Q R246A G297Q V344H L388C F014A D068L I134C N200R R246C G297R V344LL388G F014E D068P I134D N200S R246D G297S V344M L388P F014G D068V I134FN200W R246E G297T V344N L388Q F014H D068Y I134G N200Y R246G G297Y V344PL388S F014K S069N I134H G201A R246H A298C V344Q E389F F014N S069T I134KG201F R246I A298E V344R E389V F014P I070Q I134P G201L R246K A298L V344SD390A F014Q T071P I134Q G201M R246L A298M V344T D390C F014W G072C I134RG201N R246M A298N V344W D390E L015E G072F I134S G201P R246P A298P V344YD390F L015F G072H I134W G201R R246S A298Q L345A D390G L015G G072I E135PG201S R246T A298S L345C D390H L015K G072P L136P G201T R246V A298T L345ED390L L015N G072V V137F G201V R246W A298W L345H D390N L015P G072W V137GG201W V247A A298Y L345K D390P L015Q V073P V137H S202A V247C S299A L345ND390R L015R V075D V137N S202E V247F S299C L345Q D390S L015S V075G V137PS202F V247H S299D L345R D390T L015Y V075P V137R S202G V247N S299F L345TD390V W016A N076A V137W S202H V247P S299G L345V D390W W016C N076C V137YS202K V247Q S299H L345Y D390Y W016D N076F Q138V S202N V247R S299L C346AL391A W016E N076G Q139P S202P V247S S299M C346D L391D W016F N076I Q143CS202Q V247T S299P C346F L391G W016G N076K Q143H S202R V247W S299Q C346GL391H W016H N076L Q143P S202V V247Y S299T C346I L391K W016K N076P Q143RS202W R248C G300A C346K L391N W016M N076Q Q143S S202Y R248D G300C C346LL391P W016P N076R Q143T C203A R248E G300D C346M L391Q W016R N076S L144AC203D R248G G300E C346P L391R W016S N076T L144E C203E R248I G300F C346RL391S W016T N076V L144F C203G R248M G300L C346S L391T W016Y N076W L144IC203H R248P G300M C346T L391V A017D G077D L144K C203L R248T G300N C346VL391W A017E G077E L144P C203M E249A G300P C346W L391Y A017G G077L L144QC203N E249G G300Q Q347C E392C A017H G077P L144S C203Q E249H G300S Q347FE392P A017I G077Q L144V C203R E249I G300T Q347I Q393C A017L G077R L144YC203S E249K G300V Q347P Q393P A017N G077T S145T C203T E249M G300W Q347TF394A A017P G077V S145W C203V E249Q I301E Q347V F394D A017Q G078A A149EF204A E249S I301G Q347W F394E A017R G078D A149P F204C E249Y I301H E348CF394G A017S G078I T150V F204E A250C I301K E348H F394I A017T G078M K152LF204G A250F I301M E348I F394K A017V G078P A153E F204H A250G I301N E348LF394N A017W G078T A153F F204I A250H I301P E348P F394P A017Y G078Y A153MF204K A250K I301Q E348Q F394Q W018C I079A A153P F204Q A250L I301R E348RF394R W018D I079D A153R F204R A250M I301S E348T F394S W018F I079F A153TF204S A250N I301W E348V F394T W018G I079G A153V F204T A250P I301Y E348WF394V W018H I079H K154D V206C A250Q V302C E348Y S395C W018I I079K K154EV206D A250R V302D Q349D S395L W018L I079N K154G V206F A250S V302E Q349FS395M W018M I079P K154P V206G A250T V302F Q349G S395P W018P I079S K154SV206P A250V V302G Q349P E396C W018Q I079W K154W V206Y A250W V302H Q349VE396F W018S I079Y K154Y E207A I251D V302L Q349W E396G W018T P080A Q155PE207F I251F V302M Q349Y E396I W018V P080D Q155Y E207G I251G V302P G350AE396P W018Y P080E E156P E207M I251H V302R G350D E396Y N019A P080F F157AE207P I251K V302S G350E K397A N019C P080G F157C E207Q I251P V302T G350FK397C N019F P080I F157D E207R I251S V302Y G350H K397E N019G P080K F157EE207S I251T I303A G350K K397F N019H P080L F157G E207T I251W I303C G350LK397G N019I P080M F157H E207V R252A I303D G350M K397I N019L P080N F157IE207W R252D I303E G350N K397L N019M P080R F157K I208D R252E I303F G350PK397M N019P P080S F157L I208G R252F I303G G350R K397P N019Q P080T F157MI208P R252G I303K G350S K397Q N019R P080V F157P I208W R252H I303L G350TK397T N019S P080Y F157Q K209C R252I I303M G350V K397V N019V Q081A F157RK209P R252K I303R G350Y F398A N019W Q081C F157S R210A R252L I303W V351CF398C N019Y Q081E F157T R210C R252N I303Y V351D F398E A020D Q081G F157VR210D R252P W304A V351E F398G A020E Q081H E158D R210E R252S W304C V351FF398H A020F Q081L E158K R210G R252T W304D V351H F398I A020H Q081N E158PR210K R252Y W304G V351N F398L A020K Q081P E158R R210M V253A W304I V351RF398N A020L Q081S E158Y R210N V253D W304M V351W F398P A020N Q081V K159WR210P V253E W304N V351Y F398R A020P Q081W K159Y R210S V253G W304P C352AF398S A020R Q081Y G161W R210T V253H W304Q C352D F398T A020T K082W D163CR210V V253L W304S C352E F398V A020V K082Y D163P R210W V253M W304T C352FF398W A020Y I083E F164A R210Y V253N W304V C352G F398Y P021A I083K F164CN211C V253Q W304Y C352K Y399D P021C S084Y F164D N211F V253R G305L C352MY399P P021D L085A F164E N211G V253S G305P C352P C400A P021E L085C F164GN211H V253W G305Q C352Q C400D P021G L085D F164H N211I S254C G305R C352RC400E P021H L085E F164N N211K S254D G305S C352S C400F P021I L085F F164PN211M S254E G305T C352T C400G P021L L085G F164Q N211P S254G G305V C352VC400I P021M L085H F164R N211R S254I G305Y C352W C400L P021R L085N L165CN211S S254K T306A C352Y C400M P021S L085Q L165H N211T S254L T306C I353CC400P P021T L085S L165P N211V S254P T306H I353F C400Q P021V L085T L165TN211W S254Q T306I I353G C400R P021W Q086C V166D D212A S254R T306L I353HC400S S022C Q086P E167V D212G S254T T306V I353K C400T S022E D087P T168AD212H S254V T306W I353L C400V S022G H088A T168C D212I S254W T306Y I353MC400Y S022K H088C T168D D212K S254Y L307C I353Q S401C S022P H088E T168ED212L K255C I353R S401F E023A H088F T168F D212M K255D L307I I353S S401HE023F H088G T168G D212P K255L L307P I353W S401K E023L H088I T168K D212VK255P S308C R354C S401R E023M H088K T168L D212W K255V S308F R354D S401WE023N H088L T168P D213P K255W S308L R354E S401Y E023P H088M T168R D213SI256C S308M R354G C402A E023R H088P T168S L214A I256D S308V R354H C402DE023S H088R T168V L214C I256E S308W R354I C402E E023T H088S T168W L214DI256G S308Y R354K C402F E023V H088T T168Y L214E I256P M310C R354L C402LC025D H088V I169A L214G P257D M310E R354M C402M C025E H088Y I169D L214HD258L M310F R354P C402P C025F L089A I169F L214K D258P M310K R354Q C402QC025G L089D I169G L214N D258V M310L R354S C402R C025H L089E I169H L214PD258W R311C R354V C402S C025I L089G I169K L214R K260C R311E R354W C402TC025K L089Q I169N L214S K260P R311F R354Y C402V C025L L089S I169P L214TS261P R311I K355D C402W C025N L089T I169Q L214Y P262A R311L K355F C402YC025P L089W I169S S215C P262D R311P K355G Y403A C025R L089Y I169T S215PP262E R311V K355H Y403C C025S D090C I169Y W216D P262F R311W K355L Y403EC025T D090G K170C W216E P262G S312C K355M Y403G C025V K091D K170D W216GP262H S312E K355N Y403H C025Y K091E K170E W216H P262I S312M K355P Y403KG027C K091F K170G W216I P262K S312V K355Q Y403L L033C K091G K170M W216KP262Q S312W K355R Y403M L033D K091H K170P W216L P262R M313C K355S Y403NL033H K091I K170W W216M P262S K314C K355T Y403P L033N K091L K170Y W216NP262T K314L K355V Y403Q L033V K091N L171C W216P P262V K314W K355W Y403RL033Y K091T L171D W216Q P262W S315C K355Y Y403T D034I A092E L171H W216RP262Y S315I N356C S404C D034L A092F L171M W216T L263E S315V N356G S404DD034N A092H L171N W216V L263F C316E N356K S404F D034S A092K L171R L217AL263P C316G N356L S404G D034T A092P L171S L217C L263Q C316I N356P S404HD034V A092Q L171W L217G L263W C316K N356R S404L M035A A092R L171Y L217HP264D C316L N356T S404M M035D A092W G172D L217P P264E C316M N356V S404NM035G A092Y G172E L217Q P264F C316P N356W S404R M035P K094G G172I L217 SP264G C316R W357D S404V M035R K094P G172L L217T P264L C316S W357E S404WM035S D095A G172P L217V P264M C316T W357F S404Y S036C D095C G172Q L217WP264R C316V W357G T405C S036F D095E G172T W218A P264T C316W W357L T405IS036V D095F G172V W218I P264V C316Y W357M T405V S036W D095G G172W W218KP264W L317G W357Q L406P S036Y D095H G172Y W218L P264Y L317P W357R L406RL037C D095K K173D W218P V265A L318C N358E C408A L037E D095L K173E W218SV265D L318P N358H C408E L037G D095M K173G W218V V265F L318W N358I C408FL037N D095P K173H N219P V265G L319C N358K C408G L037S D095Q K173I E220GV265H L319E N358P C408I F038E D095S K173L E220K V265K L319F N358Q C408KF038G D095V K173M E220N V265L L319G N358R C408L F038K D095W K173P E220PV265M L319H N358W C408P F038L D095Y K173S E220R V265N L319I S359A C408RF038N I096A K173V E220W V265Q L319K S359F C408S F038Q I096C K173W S221DV265R L319M S359G C408T F038R I096G K173Y S221E V265S L319P S359L C408VF038T I096H L174P S221H F266A L319Q S359P C408W F038W I096P L175C S221KF266C L319R S359W C408Y S039C I096R L175D S221P F266G L319S S360A E410WS039D I096S L175G S221R F266H L319V S360C K411D S039F I096T L175K T222PF266M L319W S360E K411E S039W I096W L175P T222Y F266P L319Y S360F K411FF040A F098P L175R A223C F266Q D320C S360G K411G F040D Y099C L175S A223DF266R D320P S360I A412E F040E Y099E R176A A223E F266S D320V S360K A412HF040G Y099G R176C A223G F266T N321E S360L D413H F040K Y099I R176E A223HF266V N321M S360M D413I F040N Y099N R176F A223K F266W N321P S360P D413KF040R Y099P R176G A223L A267D Y322C S360Q D413L F040S Y099V R176H A223PA267G Y322D S360R D413P F040T Y099W R176I A223Q A267H Y322E S360V V414AF040V M100C R176P A223R A267I Y322G D361A V414D I041Q M100E R176Q A223SA267K Y322I D361C V414E G042D M100F R176S A223T A267N Y322L D361E V414GG042E M100G R176T A223V A267R Y322N D361G V414H G042H M100N R176V A223WA267S Y322P D361M V414K G042I M100P R176W A223Y A267W Y322R D361N V414RG042K M100R P177A L224A Y268A Y322S D361P V414S G042L M100S P177C L224DY268C Y322T D361Q V414T G042M M100T P177D L224E Y268F Y322V D361R K415CG042P M100W P177F L224F Y268G Y322W D361S K415D G042Q M100Y P177G L224GY268H M323A D361V K415E G042R P101A P177H L224M Y268K M323C D361W K415PG042S P101C P177L L224P Y268L M323E Y362A D416C G042T P101F P177M L224QY268N M323G Y362C D416S G042V P101H P177Q L224R Y268P M323H Y362E T417AS043A P101I P177R L224S Y268Q M323K Y362G T417D S043E P101K P177S L224TY268S M323N Y362H T417E S043F P101L P177T L224W Y268T M323R Y362K T417FS043G P101M P177V L224Y Y268V M323S Y362L T417G S043I P101N P177W Y225AY268W M323T Y362M T417H S043K P101Q N178E Y225D T269E M323V Y362N T417KS043L P101R N178I Y225E T269K E324C Y362P T417M S043Q P101S N178L Y225GT269L E324F Y362R T417P S043R P101T N178V Y225H T269M E324P Y362S T417QS043V V102P N178W Y225K T269N E324V Y362T T417R P044A D 103A N178Y Y225PT269P E324W Y362V A419D P044C D103E H179W Y225Q T269Q E324Y Y362W A419PP044F D103F L180A Y225R T269R T325C L363A V420A P044G D103G L180C Y225TR270A T325R L363C V420D P044H D103H L180E Y225V R270C I326E L363D V420FP044I D103I L180P Y225W R270E I326G L363E V420G P044L D103L L180R P226AR270F I326H L363F V420H P044N D103Q L180S P226C R270G I326N L363G V420KP044Q D103R W181A P226D R270H I326W L363H V420L P044R D103T W181C P226ER270I L327A L363I V420N P044S D103V W181D P226F R270P L327E L363P V420RP044T D103W W181E P226G R270Y L327F L363Q V420S P044W D103Y W181F P226LI271A L327G L363R V420T P044Y N104F W181H P226N I271D L327H L363S V420WR045A N104P W181I P226Q I271E L327N L363T V420Y R045D N104W W181K P226RI271H L327Q L363V V422C R045F L105C W181L P226S I271K L327R L363W V422DR045G L105M W181R P226T I271T L327S H364A V422G R045P L105N W181S P226VI271W L327T H364C V422H R045W G106A W181V P226W V272A L327V H364D V422LI046P G106C G182A P226Y V272H L327W H364E V422M I046W G106D G182C S227AV272L L327Y H364F V422N N047V G106F G182D S227F V272N P329C H364G V422QA048P G106H G182E S227G V272P P329F H364K V422R T049C G106L G182H S227HV272W P329G H364L V422S T049D G106M G182N S227I F273A P329H H364M V422YT049G G106N G182P S227K F273C P329I H364P C423A T049H G106P G182Q S227LF273D P329K H364R C423D T049P G106S G182R S227M F273G P329L H364S C423EG106W G182S S227P F273I P329N H364T C423F Q051C G106Y G182T S227Q F273LP329Q H364V C423G Q051F M107A G182V S227R F273P P329R H364Y C423H Q051IM107C G182Y S227T F273Q P329S L365A C423L Q051M M107H Y183C S227V F273SP329T L365C C423M Q051P M107K Y183D S227W F273V P329V L365D C423P Q051TM107P Y183E S227Y F273W P329W L365E C423Q Q051W M107Q Y183G I228A T274CP329Y L365G C423R Q051Y M107S Y183I I228E T274E Y330A L365M C423S G052CM107V Y183K I228F T274G Y330C L365N C423T G052E M107W Y183N I228G T274HY330D L365P C423V G052F A108D Y183P I228H T274N Y330E L365Q C423W G052WA108E Y183Q I228L T274Q Y330G L365R I424A G052Y A108F Y183R I228M T274WY330I L365S I424C V053A A108K Y183S I228N T274Y Y330L L365T I424E V053CA108L Y183V I228P D275A Y330M L365W I424G V053D A108M Y184A I228R D275FY330N L365Y I424H V053E A108P Y184C I228S D275G Y330P N366A I424N V053GA108Q Y184D I228T D275I Y330R N366C I424Q V053H A108T Y184E I228W D275KY330S N366E I424R V053L A108V Y184F Y229E D275L Y330V N366F I424S V053NA108Y Y184G Y229F D275M Y330W N366G I424W V053P V109C Y184H Y229G D275QI331A N366K I424Y V053Q V109D Y184K Y229K D275T I331C N366M A425E V053RV109E Y184L Y229L D275V I331D N366P A425L V053S V109L Y184M Y229P D275WI331E N366Q A425P V053T V109M Y184P Y229Q Q276F I331F N366R A425W V053WV109R Y184R Y229T Q276P I331H N366T A425Y V053Y V109T Y184S Y229V Q276WI331K N366W D426C T054D V109W Y184V Y229W L278M I331Q P367E D426F T054EI110F L185A L230A L278P I331R P367F D426M T054G I110K L185D L230E K279AI331S P367I D426R T054P I110L L185E L230G K279C I331T P367L G427A T054RI110M L185F L230H K279F I331W P367M G427C T054Y I110P L185G L230K K279GI331Y P367Q G427F I055A I110W L185I L230M K279L I332A P367V G427L I055DD111H L185K L230N K279W I332C D368C G427P I055G D111I L185P L230P K279YI332D D368P I055H D111Q L185R L230R F280D I332E D368W G427V I055N W112CL185S L230S F280I I332F N369C G427W I055P W112E L185T L230T F280L I332GN369E G427Y I055Q W112G L185V L230V F280M I332H N369F V428A I055R W112HL185W L230W F280N I332K N369I V428C I055T W112L L185Y L230Y F280R I332LN369K V428D I055V W112N F186A N231A F280S I332N N369L V428E I055Y W112PF186D N231C F280T I332P N369P V428G F056A W112S F186G N231D F280V I332RN369Q V428H F056C E113R F186H N231F F280W I332S N369V V428N F056E E113VF186I N231G L281A I332T N369W V428R F056G E114I F186K N231H L281D I332YF370A V428S F056H E114L F186L N231I L281G N333G F370D V428Y F056I E114PF186N N231K L281H N333H F370E C429A F056K E114T F186P N231L L281I N333IF370G C429D F056L E114V F186Q N231P L281K N333K F370H C429K F056P W115AF186R N231Q L281N N333P F370K C429L F056R W115C F186S N231R L281P N333RF370L C429N F056S W115D F186V N231S L281Q N333S F370N C429P F056T W115FF186W N231V L281R N333T F370P C429S F056V W115G P187A T232C L281S N333WF370Q C429T F056W W115H P187F T232G L281V N333Y F370R C429V Y057A W115IP187G T232H L281W V334A F370S C429W Y057D W115K P187H T232K S282F V334CF370V C429Y Y057F W115L P187I T232L S282L V334D F370Y I430A Y057G W115MP187L T232N S282V V334E A371P I430D Y057I W115R P187M T232P S282W V334GA371W I430E Y057L W115S P187N T232Q S282Y V334M I372A I430L Y057M W115VP187Q T232V Q283A V334N I372D I430M Y057P W115Y P187R T232Y Q283C V334RI372E I430N Y057Q R116A P187S Q233D Q283D V334S I372F I430S Y057R R116CP187T Q233I Q283F T335F I372G I430T Y057V R116D P187V Q233P Q283W T335GI372H I430V Y057W R116E P187W Q233S D284C T335H I372K D431P V058A R116GP187Y Q233T D284I T335I I372L A432C D059A R116H D188A Q234A D284P T335KI372N A432F D059E R1161 D188C Q234D E285K T335L I372P A432I D0591 R116LD188F Q234E E285P T335P I372R A432K D059L R116N D188G Q234G E285R T335VI372S A432L D059M R116P D188H Q234H E285T T335W I372T A432M D059P R116QD188L Q234N E285V T335Y I372V A432P D059R R116S D188M Q234P L286A L336AI372W A432Y D059T R116V D188N Q234S L286C L336E Q373C L434H D059V R116WD188P Q234T L286D L336F Q373P L434K D059W P117D D188Q Q234V L286F L336GQ373W L434P D059Y P117G D188R Q234W L286H L336K L374D L434Q R060A P117ID188S S235F L286K L336N L374E L434R R060D P117K D188T S235L L286M L336PE375C L434W R060F P117N D188V S235M L286P L336R E375F P437T R060G P117QD188W S235R L286T L336S E375P M438Y R060H P117R C189A S235W L286Y L336TE375V E439N R060I P117S C189E S235Y V287A L336V E375Y E439R R060L P117VC189G P236C V287C R121G K376I T440Q R060N P117W C189H W119L V287D R121HK376P E441R R060P T118C C189K W119N V287E R121K K376W E442M R060Q T118DC189L W119P V287G R121L G377C E442N R060S T118E C189M W119R V287K R121MG377I E442S R060T T118G C189N R121A V287L R121P G377L P443D T118R T118PT118W R121C R121F G378D G377V G378E T118Y W119I W119A W119K R121E G378FG378I

Example 5 Assay for Hyaluronidase Activity Under Temperature andPhenophilic Conditions

Supernatants from PH20 activity variants set forth in Table 9, asidentified in Example 4, were tested for stability under thermophilicand/or phenophilic conditions. The assay to measure hyaluronidaseactivity under temperature and phenophile conditions usingbiotinylated-HA (bHA) as substrate for measuring hyaluronidase activitywas modified from the original assay described in Example 3 in that itincorporated a 4-hour 37° C. incubation of samples with or withoutm-cresol prior to measurement of enzymatic activity. The assay was usedto identify PH20 mutants with thermophilic properties (activity greaterat 37° C. condition than at 4° C.) and/or with phenolphilic properties(greater activity in the presence of m-cresol than wildtype PH20).

1. Primary Screen

Prior to incubating samples with bHA, variant PH20 samples were dilutedinto designated wells of an uncoated 4×HB plate for pre-incubation at37° C. for 4 hours under the following conditions: 1) pre-incubation at37° C. with 0.4% m-cresol; and 2) pre-incubation at 37° C. without 0.4%m-cresol. For the preincubation at 37° C. with 0.4% m-cresol, a 1%m-cresol intermediate stock was prepared from 50% (v/v) m-cresol stocksolution. Briefly, in a 2 mL Wheaton glass vial a 50% stock of m-cresol(Fluka, Catalog No. 65996; Spectrum, Catalog No. C2773) was made inmethanol based on the density (D=1.034 g/L). The vial was sealed andstored at −20° C. with protection from light in small aliquotes. Then,the 1% intermediate stock was generated by dilution in HEPES assaybuffer (10 mM HEPES, 50 mM NaCl, 1 mM CaCl₂, 1 mg/mL BSA, pH 7.4, 0.05%Tween-20) daily immediately prior to use in a fume hood with vortexing.

Then, duplicates of transfected variant supernatant samples set forth inTable 9, generated as described above in Example 2, were each separatelysubjected to a 1:2.5 dilution of 1% m-cresol in HEPES assaybuffer/transfected supernatant to obtain 0.4% final concentration ofm-cresol. For the preincubation at 37° C. without 0.4% m-cresol,transfected variant supernatant samples were subjected to a 1:2.5dilution in HEPES assay buffer/transfected supernatant. In addition, foreach condition, an internal killing control was also tested by spikingin 3 U/mL of rHuPH20 in pH 7.4 HEPES buffer (generated as described inExample 1) that was diluted the same as described above for thetransfected samples. The plates were sealed with plate sealers andincubated at 37° C. for 4 hours.

The preparation of the bHA coated plates and blocking of the platesprior to addition of the transfected variant supernatants or wildtypePH20 was the same as described in Example 3. The assay was furthermodified as follows. First, samples were diluted in duplicate 1:10 inHEPES assay buffer in 4×HB plates. For each variant, the samples thatwere tested were 1) non-preincubated transfected variant supernatant (noincubation; 4° C.); 2) preincubated transfected variant supernatantspreincubated at 37° C. for 4 hours with 0.4% m-cresol (Cresol); or 3)preincubated transfected variant supernatant preincubated at 37° C. for4 hours without 0.4% m-cresol (no cresol; 37° C.). In addition, thespiked-in samples also were tested. A standard curve using rHuPH20 wasmade as described in Example 3 without m-cresol. One hundred microliters(1000) of each standard and sample were transferred to pre-designatedwells of the bHA-coated and blocked plate and incubated forapproximately 1.5 hours at 37° C. Thus, each sample of each variant wastested in quadruplicate due to the preincubation of duplicate samples ofeach transfected variant supernatants in the pre-incubation step and thefurther duplicate of each sample in the bHA assay.

After the incubation, the plates were washed and binding to bHA detectedas described above in Example 3. Optical density was measured at 450 nmwithin 30 minutes of adding the stop solution.

The U/mL activity was calculated from the standard curve and compared.The results were depicted as the percent (%) activity remaining undereach of the following parameters: ratio of activity at 1) 37° C.preincubation without m-cresol/4° C.; 2) 37° C. after preincubation withm-cresol/4° C.; and 3) 37° C. after preincubation with m-cresol/afterpreincubation at 37° C. without m-cresol. Initial phenophile hits forreconfirmation were identified as those that in a duplicate assayexhibited a percentage of remaining activity under condition 3) of ≥20%of the original activity at 37° C.

Initial Hits were rescreened using a 6-well plate rescreen assay. Forthe rescreen, plasmid DNA corresponding to the potential Hit wastransformed into E. coli bacteria and plasmid DNA prepared and purifiedusing MaxiPrep according to the manufacturers instructions. The DNAsequence was confirmed.

The plasmid DNA was transfected into monolayer CHO-S cells (Invitrogen,Cat. No. 11619-012) grown on 6-well plates at a density of about 50-80%confluency using Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027)according to the protocol suggested by the manufacturer. Transfectionswere performed in duplicate. The cells were incubated at 37° C. in a CO₂incubator for 96 hours post-transfection before collecting thesupernatant for the assay. As controls, cells also were transfected withthe HZ24-PH20(OHO)-IRES-SEAP expression vector (SEQ ID NO:4) thatcontains a codon-optimized wildtype PH20 sequence (OHO). Mock cells alsowere included as controls.

Ninety-Six (96) hours post-transfections, supernatant was collected fromeach sample, including the OHO and mock controls, and assayed forhyaluronidase activity under various conditions as described above: 1)non-preincubated transfected variant supernatant (no incubation; 4° C.);2) preincubated transfected variant supernatants preincubated at 37° C.for 4 hours with 0.4% m-cresol (Cresol; 37° C.); or 3) preincubatedtransfected variant supernatant preincubated at 37° C. for 4 hourswithout 0.4% m-cresol (no cresol; 37° C.). Hyaluronidase activity wasdetermined as described above using the bHA assay.

The results were assessed as described above. Absolute hyaluronidaseactivity (U/mL) was generated from the standard curve. In addition,percent activity was determined as a ratio of activity at 37° C./4° C.,37° C. plus m-cresol/4° C., and 37° C. plus m-cresol/37° C. The resultsare set forth in Tables 11 and 12 below.

TABLE 11 Absolute Hyaluronidase Activity No incubation 37° C. no cresol37° C. with m-cresol Mutant (4° C.) (37° C.) (37° C. plus m-cresol)L001A 2.993 2.511 3.529 3.214 0.287 0.295 L001E 2.669 2.539 2.862 3.1790.376 0.341 L001G 0.348 0.583 0.596 0.676 0.055 0.031 L001Q 5.135 6.4436.133 5.719 0.621 0.636 L001R 5.603 4.390 6.576 7.042 0.458 0.396 P006A2.965 3.208 4.088 3.495 0.404 0.435 V008M 1.376 1.401 1.856 1.678 0.0000.008 I009Q 0.447 0.381 0.469 0.476 0.031 0.030 P010G 0.747 0.564 0.8200.688 0.123 0.114 P010H 0.473 0.485 0.624 0.548 0.000 0.000 N011S 0.8620.962 1.313 1.263 0.094 0.064 V012E 11.019 5.519 5.312 5.528 0.753 0.934V012I 2.804 3.844 3.610 6.566 0.106 0.090 V012K 1.691 1.963 2.479 2.2430.330 0.321 F014V 0.144 0.165 0.222 0.242 0.003 0.000 L015M 0.902 1.0731.026 0.901 0.017 0.017 A020S 1.494 2.205 2.822 2.620 0.413 0.397 S022T3.035 3.788 3.375 3.273 0.684 0.748 L026M 1.482 1.226 2.027 1.704 0.2240.178 K028R 0.944 0.845 1.043 0.925 0.112 0.095 F029R 1.195 1.511 1.8481.839 0.140 0.140 F029S 3.019 3.615 3.566 3.521 0.250 0.283 F029T 1.4511.712 1.839 2.065 0.220 0.212 P032C 0.370 0.419 0.476 0.534 0.006 0.040L033G 0.566 0.700 0.686 0.627 0.001 0.026 D034W 0.340 0.321 0.499 0.4710.076 0.069 M035V 0.887 0.639 0.721 0.652 0.116 0.023 S036H 1.109 0.7521.178 1.135 0.117 0.026 S036N 0.797 0.933 0.893 0.859 0.171 0.260 L037M0.574 0.404 0.455 0.353 0.049 0.032 F040L 2.603 3.941 3.515 4.148 0.2770.361 I046L 3.027 2.959 4.011 3.342 0.513 0.557 N047D 2.222 2.359 2.5732.639 0.032 0.021 N047W 0.404 0.415 0.423 0.456 0.000 0.017 A048N 12.39845.971 14.252 23.873 0.797 0.902 T049R 7.893 13.334 9.685 12.102 0.5630.649 G050D 3.287 3.148 3.084 3.020 0.242 0.264 G050M 1.763 2.333 2.7803.244 0.250 0.393 G052N 7.217 9.809 6.939 13.978 1.109 1.083 G052T 1.5421.224 1.795 1.433 0.381 0.463 G052S 2.152 1.999 2.120 1.963 0.498 0.566V058C 1.428 1.312 1.321 1.301 0.212 0.210 V058K 28.000 28.000 61.01661.016 23.586 23.586 V058R 5.719 4.688 5.542 4.822 3.134 3.149 V058N1.200 1.175 1.550 1.525 0.200 0.175 V058Y 1.040 0.770 1.071 1.088 0.3880.454 V058Q 11.956 15.363 18.458 45.092 1.567 2.166 V058P 3.360 2.9492.799 5.121 0.592 0.884 V058H 3.790 5.074 7.590 9.222 0.826 1.205 D068P0.215 0.215 0.213 0.180 0.001 0.184 S069T 1.927 2.179 2.671 2.671 0.2890.240 I070P 1.284 1.593 1.306 1.589 0.010 0.032 I070V 1.818 2.437 3.0993.335 0.433 0.363 V073Q 4.846 5.441 5.880 5.827 0.383 0.477 V073R 0.5220.803 0.720 0.804 0.018 0.059 T074E 2.903 3.834 3.868 3.871 0.666 0.626T074M 0.569 0.744 0.656 0.771 0.079 0.083 T074N 2.792 1.905 2.565 2.9950.281 0.204 T074P 2.331 1.593 2.525 2.648 0.309 0.265 T074R 0.999 0.8200.806 1.066 0.060 0.023 T074V 1.186 1.280 1.365 1.460 0.101 0.080 V075M0.917 1.087 1.233 1.321 0.003 0.028 K082L 1.362 1.311 1.563 3.302 0.3250.354 K082N 3.202 3.411 3.396 3.244 0.792 0.861 I083V 3.706 2.633 5.1943.615 1.552 1.017 I083Q 2.376 1.946 2.665 3.674 0.720 0.510 I083S 0.8411.054 0.880 1.005 0.235 0.268 I083G 2.276 2.443 2.418 1.866 0.545 0.601S084E 1.470 1.484 1.834 1.683 0.115 0.115 S084F 1.179 1.212 0.982 1.1030.025 0.000 S084N 2.255 1.888 3.268 2.476 0.597 0.547 S084R 8.534 14.77910.230 30.016 1.117 1.494 Q086A 2.084 2.120 2.845 3.310 0.405 0.322Q086H 1.187 1.000 1.218 1.296 0.087 0.065 Q086K 0.127 0.110 0.126 0.0720.032 0.023 Q086S 2.528 2.082 2.539 2.149 0.173 0.241 Q086T 3.018 2.5422.832 4.562 0.290 0.406 D087G 2.755 2.176 2.252 1.971 0.034 0.122 D087L2.070 2.277 2.195 2.311 0.324 0.299 D087M 2.262 2.325 2.510 2.038 0.1910.335 D087S 5.210 10.305 6.983 14.399 0.569 0.928 D087V 1.361 1.3641.553 1.187 0.142 0.189 D090E 8.251 12.299 7.666 19.836 1.093 1.234D090N 2.812 2.775 3.123 2.737 0.379 0.290 K093Q 2.491 2.065 2.267 1.9710.132 0.131 K093R 2.986 2.862 3.094 2.842 0.362 0.465 K094D 2.393 2.0882.071 2.132 0.135 0.211 K094R 1.407 1.542 1.764 1.676 0.158 0.166 T097C0.330 0.618 0.545 0.505 0.044 0.087 T097D 0.520 0.565 0.643 0.664 0.0550.073 T097E 1.096 1.410 1.394 1.623 0.217 0.262 T097L 0.899 1.198 1.0651.241 0.246 0.300 N104R 2.508 2.356 2.876 2.790 0.279 0.238 A120H 2.1552.551 2.028 2.883 0.168 0.199 D127R 0.264 0.339 0.149 0.199 0.105 0.068V128I 3.120 3.313 3.546 3.401 0.389 0.504 N131M 15.335 20.678 27.14315.899 0.505 0.447 N131R 8.195 8.748 7.724 8.392 1.645 1.626 N131V 1.6561.870 2.280 1.962 0.233 0.214 R132L 3.306 3.235 3.259 2.966 0.337 0.430Q138L 1.494 1.660 1.611 1.521 0.410 0.347 Q140K 2.829 4.065 4.996 4.4640.546 0.559 N141R 1.290 1.320 1.334 1.527 0.058 0.035 N141S 2.201 2.7082.900 2.966 0.135 0.164 N141W 1.475 1.568 1.927 1.643 0.100 0.105 V142D2.552 2.186 2.914 3.193 0.128 0.067 V142G 1.357 1.796 1.597 1.621 0.2110.219 V142K 3.532 2.381 3.867 3.681 0.571 0.575 V142N 0.432 0.567 0.6720.589 0.103 0.087 V142P 4.624 7.213 7.722 7.021 1.074 1.081 V142Q 5.0906.900 7.618 6.897 0.678 0.678 V142R 1.968 2.595 2.941 2.689 0.364 0.330V142S 2.789 2.988 4.763 3.497 0.416 0.591 V142T 1.926 3.260 4.313 4.0310.495 0.472 Q143G 3.922 4.903 5.632 4.846 0.782 0.780 Q143K 3.634 3.6717.285 5.008 1.043 1.039 L144R 3.810 4.581 5.191 5.107 0.556 0.520 L144T1.496 1.681 1.941 1.831 0.285 0.219 L146P 0.818 0.782 0.954 0.904 0.0110.031 T147S 0.984 1.149 1.399 1.497 0.055 0.039 T150N 0.442 0.585 0.6220.684 0.039 0.046 T150S 1.747 1.400 1.875 1.988 0.120 0.121 E151A 2.8702.269 2.965 2.860 0.359 0.337 E151L 3.365 3.289 4.446 4.007 0.218 0.251E151S 5.187 4.591 5.987 6.262 0.371 0.294 E151T 2.442 3.000 3.134 3.3090.000 0.000 E151V 3.998 4.247 4.459 4.232 0.326 0.314 E151W 7.166 14.24811.352 13.524 0.131 0.121 K152T 1.204 1.377 1.796 1.883 0.100 0.067K152W 2.084 1.795 2.549 2.406 0.063 0.069 E158S 0.339 0.397 0.451 0.4070.000 0.000 K162E 0.168 0.195 0.114 0.080 0.004 0.024 L165F 4.775 5.2505.075 5.075 0.600 0.725 V166Q 1.883 2.507 2.937 2.958 0.392 0.324 V166T0.993 1.315 1.821 1.800 0.231 0.235 E167D 0.811 0.910 1.109 1.480 0.1110.056 I169L 1.812 1.796 2.540 2.196 0.335 0.341 K170R 1.578 2.054 2.5361.995 0.209 0.201 G172A 0.413 0.581 0.692 0.777 0.052 0.056 K173R 1.6541.551 1.766 2.083 0.173 0.156 L174G 0.184 0.087 0.210 0.230 0.026 0.031L174N 1.616 2.276 2.494 2.872 0.331 0.543 L174T 0.552 0.566 0.689 0.8200.090 0.050 N178K 2.931 4.375 4.891 4.513 0.258 0.362 N178R 8.160 13.82016.287 20.033 0.665 0.790 H193Q 1.060 1.367 2.264 1.888 0.346 0.346K195T 1.227 0.806 1.548 1.911 0.348 0.292 K195N 1.266 1.437 1.649 1.3850.369 0.353 K196E 0.732 0.660 0.663 1.017 0.244 0.239 K196R 2.246 2.2852.383 2.174 0.315 0.384 F204P 3.500 4.550 2.925 3.750 2.475 4.725 N205A0.515 0.837 0.717 0.854 0.153 0.160 N205E 1.011 2.004 1.627 1.870 0.3140.346 N205L 1.084 1.029 1.165 0.000 0.123 0.088 N205T 0.295 0.367 0.4280.406 0.043 0.053 V206I 0.317 0.508 0.600 0.565 0.079 0.088 K209R 2.0412.453 2.445 1.951 0.291 0.077 D212N 5.568 4.549 6.271 6.016 0.167 0.322D212S 1.987 1.502 2.442 2.222 0.204 0.152 D213A 0.235 0.283 0.432 0.4380.116 0.060 D213M 1.664 2.080 2.650 2.046 0.181 0.142 S215H 2.448 3.0562.670 2.414 0.268 0.139 S215M 1.497 2.175 2.618 1.630 0.110 0.146 N219I0.338 0.250 0.860 0.728 0.076 0.082 E220V 3.783 3.828 4.993 4.349 0.3710.257 T222G 3.528 5.262 5.399 5.549 0.033 0.044 T232F 0.539 1.242 0.7160.781 0.089 0.153 Q233G 0.041 0.095 0.115 0.121 0.000 0.000 Q234M 6.0296.031 5.764 4.871 1.286 0.988 S235A 0.550 0.502 0.714 0.607 0.079 0.073V237C 0.623 0.708 0.860 0.824 0.000 0.000 V237H 0.303 0.316 0.370 0.4590.046 0.034 V237T 0.152 0.196 0.254 0.247 0.054 0.053 A238E 2.050 1.8001.945 2.559 0.159 0.171 A238H 0.579 0.363 0.345 0.743 0.090 0.062 T240A1.107 0.900 1.564 1.302 0.143 0.118 T240Q 0.333 0.510 0.542 0.617 0.0800.085 R248A 2.274 2.499 2.575 3.115 0.027 0.075 E249V 3.001 3.894 4.2844.325 0.655 0.712 P257G 3.981 4.452 4.985 5.022 0.039 0.034 K260M 0.7190.960 0.839 0.935 0.072 0.068 S261A 3.253 3.117 1.872 2.686 1.264 1.451S261K 6.089 5.421 9.860 6.297 1.583 1.437 S261N 14.149 40.257 20.21914.303 2.115 1.917 A267T 0.052 0.095 0.102 0.106 0.036 0.041 F273H 0.3400.436 0.417 0.519 0.025 0.031 F273Y 0.558 0.505 0.668 0.519 0.052 0.050Q276H 2.706 1.877 2.027 1.997 0.181 0.201 Q276M 0.775 0.768 0.762 0.8060.043 0.000 Q276R 6.080 9.717 7.383 14.593 0.807 1.281 Q276S 1.353 1.2121.497 1.681 0.149 0.147 V277A 1.202 1.643 1.692 2.129 0.118 0.110 V277E2.440 2.340 4.289 4.577 0.161 0.239 V277H 5.548 5.302 7.181 7.300 0.2270.512 V277K 8.950 8.996 33.627 33.627 4.442 4.045 V277M 1.279 1.6221.754 1.818 0.264 0.270 V277N 14.351 4.306 12.865 11.772 0.938 0.796V277Q 5.459 5.461 6.547 6.343 0.373 0.493 V277R 18.300 12.038 17.58120.641 2.737 2.023 V277S 14.351 10.444 9.509 15.135 0.727 0.716 V277T8.412 7.804 8.497 11.184 0.679 0.871 L278E 4.416 2.795 3.330 2.800 0.1700.202 L278G 7.502 7.456 9.173 7.760 0.596 0.612 K279H 0.888 1.087 1.2341.339 0.185 0.269 V287T 0.580 0.667 0.843 0.832 0.139 0.100 T289S 0.7831.019 0.819 1.001 0.008 0.007 G291S 0.227 0.322 0.419 0.385 0.051 0.016G291V 3.662 3.707 4.131 5.599 0.821 0.706 E292C 1.344 1.599 1.711 1.6170.138 0.144 E292F 6.106 4.697 8.422 6.216 0.520 0.363 E292H 2.620 3.3164.458 3.830 0.389 0.451 E292R 2.810 2.178 3.155 2.829 0.398 0.339 E292V0.891 1.121 1.453 1.494 0.193 0.177 T293A 1.986 3.110 2.546 1.789 0.0860.076 A298G 0.161 0.274 0.342 0.236 0.030 0.022 L307G 0.616 0.661 0.7260.605 0.000 0.000 S308D 0.264 0.325 0.337 0.344 0.014 0.010 S308K 0.6510.722 0.826 0.716 0.011 0.000 S308N 3.995 4.406 6.808 6.128 0.386 0.362I309E 3.166 2.819 3.921 3.663 0.637 0.528 I309G 6.651 5.429 6.824 6.1940.503 0.400 I309L 0.326 0.403 0.501 0.431 0.048 0.047 I309M 2.809 2.4733.467 3.383 0.278 0.239 I309N 4.865 5.191 5.444 5.054 0.380 0.327 I309S10.719 28.759 18.217 158.604 0.748 1.367 I309T 3.052 2.509 2.989 3.7350.228 0.207 I309V 1.705 1.292 1.929 1.787 0.029 0.062 M310G 4.514 6.3977.568 7.084 0.866 0.915 M310Q 3.648 3.179 3.912 3.380 1.088 0.955 M313G0.252 0.325 0.348 0.355 0.034 0.036 M313H 3.767 5.276 10.243 10.3950.380 0.404 M313K 12.689 12.122 15.085 12.984 0.129 0.072 M313P 4.0502.951 4.198 3.919 0.209 0.177 M313R 4.634 10.863 7.288 3.568 0.337 0.296M313T 2.903 4.474 4.705 4.467 0.331 0.313 M313Y 1.063 1.262 1.276 1.3000.096 0.089 K314S 2.848 4.450 4.042 5.879 0.391 0.533 K314Y 0.093 0.1310.226 0.182 0.013 0.020 S315A 1.472 1.082 1.345 1.484 0.222 0.148 S315H2.412 3.242 3.648 3.414 0.440 0.371 S315Y 0.279 0.626 0.477 0.362 0.1460.143 L317A 3.254 2.845 4.019 3.776 0.280 0.317 L317I 1.078 1.524 2.0211.687 0.257 0.180 L317K 12.129 9.382 11.668 12.591 0.402 0.445 L317N2.907 3.066 3.703 3.717 0.445 0.540 L317R 8.631 15.187 20.585 15.1060.796 0.857 L317S 11.586 29.267 10.535 25.114 1.637 1.613 L317T 1.3381.073 1.953 1.656 0.136 0.018 L317W 0.810 1.128 1.326 1.665 0.158 0.171L318D 1.750 1.970 1.847 1.930 0.322 0.322 L318H 1.073 0.806 1.072 1.0050.046 0.074 L318R 2.856 3.464 4.583 4.187 0.258 0.260 N321R 3.069 4.4095.059 4.946 0.482 0.426 N321S 0.683 0.710 0.700 0.772 0.058 0.035 E324N4.309 2.530 4.508 3.321 0.348 0.303 T325E 1.071 1.270 1.337 1.352 0.1930.143 N328G 0.379 0.504 0.747 0.553 0.031 0.040 N328Y 2.629 4.543 4.7584.543 0.490 0.477 T335S 0.905 0.787 0.977 0.986 0.113 0.062 Q347A 8.31611.961 8.432 11.508 0.918 1.266 Q347G 1.358 1.120 3.021 2.319 0.2530.209 Q349M 1.493 1.629 1.486 1.760 0.178 0.217 Q349R 0.451 0.572 0.6630.598 0.078 0.079 V351S 1.379 1.633 1.804 1.647 0.000 0.000 I353V 2.3351.954 3.090 2.697 0.323 0.321 N356H 0.445 0.451 0.445 0.588 0.038 0.023N356S 0.262 0.253 0.136 0.318 0.000 0.008 S359E 2.616 2.635 3.547 3.5600.382 0.333 S359H 0.403 0.371 0.445 0.374 0.000 0.000 P367A 0.643 0.7821.074 0.996 0.139 0.131 P367G 0.593 0.530 0.686 0.650 0.000 0.000 P367K0.707 0.767 0.890 0.513 0.045 0.052 P367S 3.967 3.478 2.946 3.073 0.4240.505 D368A 1.762 2.321 2.143 1.895 0.031 0.040 D368E 3.464 4.944 5.7724.842 0.530 0.555 D368L 0.557 0.566 0.607 0.619 0.000 0.006 D368M 0.8611.065 1.031 1.104 0.028 0.028 D368R 4.503 5.270 7.418 6.226 0.754 0.735D368T 2.345 1.993 2.512 2.525 0.072 0.085 N369R 1.548 2.719 2.503 2.0220.160 0.125 A371F 2.760 5.207 4.974 3.980 0.308 0.222 A371H 8.101 86.58777.531 77.531 1.403 1.316 A371H 3.509 4.058 3.900 3.879 0.000 0.334A371K 2.903 3.546 3.963 4.055 0.509 0.505 A371L 11.018 40.668 76.58743.516 1.159 0.964 A371L 3.328 3.445 3.472 2.075 0.000 0.025 A371R25.855 25.855 n/a n/a 2.851 3.634 A371R 6.592 7.733 7.987 7.576 0.0000.196 A371S 3.329 3.505 4.916 4.611 0.412 0.781 L374P 2.939 7.129 11.5228.771 0.665 0.646 E375A 0.627 0.507 0.557 0.683 0.000 0.014 E375G 1.5961.299 2.025 1.806 0.209 0.265 E375R 0.937 1.132 1.529 1.318 0.201 0.260K376D 0.458 0.312 0.518 0.515 0.064 0.026 K376E 1.572 1.094 1.572 1.6740.213 0.174 K376Q 0.727 0.940 0.910 0.846 0.116 0.102 K376R 2.086 1.3511.704 2.690 0.539 0.279 K376T 0.847 1.001 1.026 1.135 0.153 0.064 K376V0.834 0.861 1.036 1.021 0.033 0.026 K376Y 1.316 0.777 1.353 0.747 0.1250.097 G377D 1.159 1.332 1.285 1.763 0.202 0.186 G377E 0.877 0.926 1.1441.189 0.092 0.088 G377H 3.037 3.432 4.460 3.598 0.372 0.364 G377K 3.4454.101 6.405 4.911 0.283 0.245 G377R 1.096 1.257 1.312 1.191 0.077 0.085G377S 0.453 0.452 0.492 0.457 0.034 0.036 G377T 2.198 2.313 2.474 2.5220.424 0.461 F380W 17.497 27.987 25.734 29.353 2.566 2.716 T381S 2.8613.161 3.886 3.558 0.521 0.367 R383I 1.959 6.936 10.340 6.820 0.655 0.513R383S 2.429 2.548 3.228 3.044 0.339 0.321 K385A 0.479 0.669 0.604 0.7540.028 0.000 K385Q 1.746 2.089 2.403 2.609 0.217 0.196 K385V 1.232 1.7501.387 1.410 0.071 0.042 E389A 6.872 10.944 21.081 24.610 0.449 0.449E389G 0.166 0.203 0.188 0.284 0.004 0.000 E389L 1.814 2.142 2.598 2.4030.370 0.303 E389Q 2.547 3.432 3.459 3.423 0.411 0.437 E389S 1.847 2.6403.059 2.456 0.000 0.007 E392A 1.797 1.370 2.021 2.133 0.147 0.136 E392F1.575 1.407 1.821 2.023 0.071 0.079 E392Q 5.826 4.653 6.583 4.364 0.6930.729 E392R 4.555 5.306 5.900 6.548 0.218 0.193 E392V 3.817 2.936 4.7474.544 0.367 0.291 Q393F 1.754 2.186 2.455 2.222 0.260 0.226 Q393M 1.2521.826 1.749 1.588 0.028 0.049 S395A 4.220 6.127 8.788 6.906 1.141 0.856S395H 1.609 2.261 2.574 2.564 0.323 0.268 E396A 1.135 1.184 1.497 1.5240.126 0.149 E396H 0.357 0.532 0.751 0.684 0.069 0.022 E396Q 1.310 1.6251.611 1.559 0.162 0.160 E396S 3.375 5.709 5.274 6.380 0.146 0.129 Y399T2.538 3.250 3.313 3.989 0.000 0.002 Y399V 2.738 2.697 3.028 3.129 0.4840.557 Y399W 1.400 1.883 1.715 1.946 0.236 0.233 S401A 2.636 3.171 3.2163.148 0.447 0.410 S401E 1.685 1.601 2.110 2.060 0.344 0.309 S404A 1.2881.635 1.924 1.724 0.000 0.019 L406F 0.706 0.490 0.867 0.716 0.000 0.000L406N 0.617 0.795 0.943 1.044 0.060 0.070 S407A 2.428 2.949 3.432 3.2550.389 0.548 S407D 2.090 5.790 5.038 5.682 0.569 0.575 S407P 2.660 2.7083.812 3.301 0.261 0.366 A412Q 2.001 2.918 2.925 2.902 0.279 0.247 A412R4.562 5.132 6.390 6.347 0.570 0.596 A412V 2.581 3.451 3.789 3.511 0.1890.189 D416L 0.610 0.817 0.737 1.043 0.130 0.160 D418R 4.541 4.847 5.3475.438 0.406 0.583 A419H 10.409 20.311 25.109 38.221 2.214 2.293 A419K12.835 10.298 24.536 208.289 2.556 3.173 D421A 5.968 5.617 6.094 16.9400.761 0.764 D421H 48.012 48.012 160.106 32.481 16.300 28.113 D421K 5.5275.225 6.864 5.346 0.523 0.725 D421N 9.060 8.635 10.039 8.645 1.502 1.422D421Q 7.529 5.581 7.858 8.016 0.842 0.994 D421R 6.637 5.463 9.211 7.5370.815 0.737 D421S 5.556 5.355 7.899 8.898 0.869 0.762 A425G 10.421 8.8277.796 10.676 0.827 1.189 G427Q 1.008 1.252 1.342 1.230 0.031 0.106 G427T1.330 1.380 1.664 1.643 0.080 0.065 V428L 2.138 2.769 2.930 3.029 0.0530.030 D431E 2.810 2.220 1.972 2.112 0.519 0.438 D431H 2.154 3.185 4.0173.028 0.294 0.301 D431K 8.123 16.953 19.563 11.575 2.272 2.339 D431L1.211 1.215 1.564 1.448 0.164 0.170 D431N 11.819 12.063 16.358 15.1311.601 1.399 D431Q 6.077 9.828 14.157 10.760 1.533 1.153 D431S 14.52310.220 11.338 9.075 0.853 0.829 F433A 4.035 4.673 5.943 4.649 0.5810.595 F433H 1.836 2.397 2.574 2.108 0.347 0.356 F433I 2.754 2.643 2.9902.299 0.338 0.382 F433K 17.815 14.495 16.240 49.615 1.806 1.790 F433R8.198 6.719 10.572 8.960 1.113 0.857 F433T 6.005 5.941 9.716 8.019 1.3271.542 F433V 10.645 7.762 150.315 8.696 2.415 1.505 F433W 0.526 0.7950.784 0.903 0.082 0.068 P437I 0.759 0.996 1.130 1.066 0.027 0.019 M438A1.996 1.518 2.125 2.060 0.214 0.210 M438D 2.849 2.522 3.002 2.857 0.3050.074 M438E 4.681 4.992 5.386 5.680 0.431 0.518 M438L 10.127 5.268 6.66311.324 0.670 0.739 M438N 6.172 5.531 8.050 5.568 0.649 0.662 M438T 2.2182.411 2.308 2.500 0.309 0.304 E439A 3.557 4.432 4.883 4.235 0.568 0.596E439A 1.099 0.998 1.694 1.470 0.080 0.109 E439C 0.148 0.256 0.286 0.2860.042 0.045 E439K 0.466 0.588 0.580 0.616 0.077 0.065 E439P 2.868 3.7363.394 3.267 0.529 0.490 E439Q 1.070 0.848 1.087 1.080 0.116 0.115 E439T1.965 1.889 2.179 2.323 0.313 0.263 T440D 4.148 4.443 4.931 3.533 0.5680.651 T440H 2.317 1.982 3.297 2.595 0.147 0.196 T440M 3.397 3.305 2.8782.873 0.254 0.367 T440P 3.562 3.593 3.987 3.277 0.540 0.566 T440S 2.5222.207 2.533 2.895 0.283 0.284 E441F 1.402 1.407 1.813 1.560 0.204 0.178E442G 2.871 3.340 3.193 3.347 0.327 0.367 P443E 0.907 0.710 0.856 0.9280.044 0.063 P443F 1.830 2.370 2.683 2.321 0.301 0.286 P443G 4.077 2.9219.751 4.614 0.835 0.756 Q444E 8.293 3.861 6.800 6.213 0.581 0.594 Q444H3.823 3.936 5.746 4.710 0.486 0.513 Q444V 2.193 2.107 2.847 2.583 0.3840.284 I445M 5.265 4.438 4.480 4.489 0.773 0.691 I445N 3.375 4.024 3.5923.515 0.499 0.455 I445W 2.289 2.694 2.683 2.695 0.314 0.296 Y447E 2.3732.464 2.363 2.685 0.391 0.345 Y447G 0.945 1.352 1.358 1.401 0.187 0.162Y447P 0.991 1.383 1.379 1.490 0.190 0.183 positive 2.919 2.173 2.7732.105 0.145 0.178 control 3.984 4.463 4.215 4.823 0.189 0.253 (OHO) 32.725 3 3.325 0.1 0.125 2.501 2.883 2.370 3.158 0.452 0.522 7.629 2.98910.835 3.914 0.485 0.219 5.783 5.356 2.609 3.643 0.542 0.402 5.279 5.4222.815 4.026 0.618 0.401 4.775 4.385 2.845 3.327 0.718 0.540 3.617 4.2643.322 3.427 0.633 0.479 5.881 4.511 5.518 4.359 0.743 0.848 6.754 4.9323.902 4.120 0.665 0.724 3.911 3.494 3.911 5.179 0.726 0.841 5.406 7.5594.018 4.620 0.735 0.429 4.015 3.887 3.9400 3.4080 0.3340 0.3410 2.6042.339 2.4430 2.3910 0.2350 0.2330 3.736 3.473 3.6210 3.0560 0.31000.2770 3.759 3.509 3.6330 3.0490 0.3600 0.3030 n/a (not available; e.g.,beyond detection limit)

TABLE 12 Percent (%) Activity duplicate 1 duplicate 2 % activity %activity % activity % activity % activity % activity at 37° C. + m- 37°C. + m- at 37° C. + m- 37° C. + m- 37° C./4° C. cresol/37° C. cresol/4°C. 37° C./4° C. cresol/37° C. cresol/4° C. L001A 117.908 8.13 9.59127.997 9.179 11.75 L001E 107.231 13.14 14.09 125.207 10.727 13.43 L001G171.264 9.23 15.80 115.952 4.586 5.32 L001Q 119.435 10.13 12.09 88.76311.121 9.87 L001R 117.366 6.96 8.17 160.410 5.623 9.02 P006A 137.8759.88 13.63 108.946 12.446 13.56 V008M 134.884 0.00 0.00 119.772 0.4770.57 I009Q 104.922 6.61 6.94 124.934 6.303 7.87 P010G 109.772 15.0016.47 121.986 16.570 20.21 P010H 131.924 0.00 0.00 112.990 0.000 0.00N011S 152.320 7.16 10.90 131.289 5.067 6.65 V012E 48.208 14.18 6.83100.163 16.896 16.92 V012I 128.745 2.94 3.78 170.812 1.371 2.34 V012K146.600 13.31 19.52 114.264 14.311 16.35 F014V 154.167 1.35 2.08 146.6670.000 0.00 L015M 113.747 1.66 1.88 83.970 1.887 1.58 A020S 188.889 14.6427.64 118.821 15.153 18.00 S022T 111.203 20.27 22.54 86.404 22.854 19.75L026M 136.775 11.05 15.11 138.989 10.446 14.52 K028R 110.487 10.74 11.86109.467 10.270 11.24 F029R 154.644 7.58 11.72 121.707 7.613 9.27 F029S118.119 7.01 8.28 97.400 8.037 7.83 F029T 126.740 11.96 15.16 120.61910.266 12.38 P032C 128.649 1.26 1.62 127.446 7.491 9.55 L033G 121.2010.15 0.18 89.571 4.147 3.71 D034W 146.765 15.23 22.35 146.729 14.65021.50 M035V 81.285 16.09 13.08 102.034 3.528 3.60 S036H 106.222 9.9310.55 150.931 2.291 3.46 S036N 112.045 19.15 21.46 92.069 30.268 27.87L037M 79.268 10.77 8.54 87.376 9.065 7.92 F040L 135.036 7.88 10.64105.252 8.703 9.16 I046L 132.507 12.79 16.95 112.944 16.667 18.82 N047D115.797 1.24 1.44 111.869 0.796 0.89 N047W 104.703 0.00 0.00 109.8803.728 4.10 A048N 114.954 5.59 6.43 51.931 3.778 1.96 T049R 122.704 5.817.13 90.760 5.363 4.87 G050D 93.824 7.85 7.36 95.934 8.742 8.39 G050M157.686 8.99 14.18 139.048 12.115 16.85 G052N 96.148 15.98 15.37 142.5027.748 11.04 G052T 116.407 21.23 24.71 117.075 32.310 37.83 G052S 98.51323.49 23.14 98.199 28.833 28.31 V058C 92.507 16.05 14.85 99.162 16.14116.01 V058K 217.914 38.66 84.24 217.914 38.655 84.24 V058R 96.905 56.5554.80 102.858 65.305 67.17 V058N 129.167 12.90 16.67 129.787 11.47514.89 V058Y 102.981 36.23 37.31 141.299 41.728 58.96 V058Q 154.383 8.4913.11 293.510 4.804 14.10 V058P 83.304 21.15 17.62 173.652 17.262 29.98V058H 200.264 10.88 21.79 181.750 13.067 23.75 D068P 99.070 0.47 0.4783.721 102.222 85.58 S069T 138.609 10.82 15.00 122.579 8.985 11.01 I070P101.713 0.77 0.78 99.749 2.014 2.01 I070V 170.462 13.97 23.82 136.84910.885 14.90 V073Q 121.337 6.51 7.90 107.094 8.186 8.77 V073R 137.9312.50 3.45 100.125 7.338 7.35 T074E 133.241 17.22 22.94 100.965 16.17216.33 T074M 115.290 12.04 13.88 103.629 10.765 11.16 T074N 91.870 10.9610.06 157.218 6.811 10.71 T074P 108.323 12.24 13.26 166.227 10.008 16.64T074R 80.681 7.44 6.01 130.000 2.158 2.80 T074V 115.093 7.40 8.52114.063 5.479 6.25 V075M 134.460 0.24 0.33 121.527 2.120 2.58 K082L114.758 20.79 23.86 251.869 10.721 27.00 K082N 106.059 23.32 24.7395.104 26.541 25.24 I083V 140.151 29.88 41.88 137.296 28.133 38.63 I083Q112.163 27.02 30.30 188.798 13.881 26.21 I083S 104.637 26.70 27.9495.351 26.667 25.43 I083G 106.239 22.54 23.95 76.381 32.208 24.60 S084E124.762 6.27 7.82 113.410 6.833 7.75 S084F 83.291 2.55 2.12 91.007 0.0000.00 S084N 144.922 18.27 26.47 131.144 22.092 28.97 S084R 119.873 10.9213.09 203.099 4.977 10.11 Q086A 136.516 14.24 19.43 156.132 9.728 15.19Q086H 102.612 7.14 7.33 129.600 5.015 6.50 Q086K 99.213 25.40 25.2065.455 31.944 20.91 Q086S 100.435 6.81 6.84 103.218 11.215 11.58 Q086T93.837 10.24 9.61 179.465 8.900 15.97 D087G 81.742 1.51 1.23 90.5796.190 5.61 D087L 106.039 14.76 15.65 101.493 12.938 13.13 D087M 110.9647.61 8.44 87.656 16.438 14.41 D087S 134.031 8.15 10.92 139.728 6.4459.01 D087V 114.107 9.14 10.43 87.023 15.922 13.86 D090E 92.910 14.2613.25 161.281 6.221 10.03 D090N 111.060 12.14 13.48 98.631 10.596 10.45K093Q 91.008 5.82 5.30 95.448 6.646 6.34 K093R 103.617 11.70 12.1299.301 16.362 16.25 K094D 86.544 6.52 5.64 102.107 9.897 10.11 K094R125.373 8.96 11.23 108.690 9.905 10.77 T097C 165.152 8.07 13.33 81.71517.228 14.08 T097D 123.654 8.55 10.58 117.522 10.994 12.92 T097E 127.19015.57 19.80 115.106 16.143 18.58 T097L 118.465 23.10 27.36 103.58924.174 25.04 N104R 114.673 9.70 11.12 118.421 8.530 10.10 A120H 94.1078.28 7.80 113.015 6.903 7.80 D127R 56.439 70.47 39.77 58.702 34.17120.06 V128I 113.654 10.97 12.47 102.656 14.819 15.21 N131M 177.000 1.863.29 76.888 2.811 2.16 N131R 94.253 21.30 20.07 95.930 19.376 18.59N131V 137.681 10.22 14.07 104.920 10.907 11.44 R132L 98.578 10.34 10.1991.685 14.498 13.29 Q138L 107.831 25.45 27.44 91.627 22.814 20.90 Q140K176.600 10.93 19.30 109.815 12.522 13.75 N141R 103.411 4.35 4.50 115.6822.292 2.65 N141S 131.758 4.66 6.13 109.527 5.529 6.06 N141W 130.644 5.196.78 104.783 6.391 6.70 V142D 114.185 4.39 5.02 146.066 2.098 3.06 V142G117.686 13.21 15.55 90.256 13.510 12.19 V142K 109.485 14.77 16.17154.599 15.621 24.15 V142N 155.556 15.33 23.84 103.880 14.771 15.34V142P 166.998 13.91 23.23 97.338 15.397 14.99 V142Q 149.666 8.90 13.3299.957 9.830 9.83 V142R 149.441 12.38 18.50 103.622 12.272 12.72 V142S170.778 8.73 14.92 117.035 16.900 19.78 V142T 223.936 11.48 25.70123.650 11.709 14.48 Q143G 143.600 13.88 19.94 98.837 16.096 15.91 Q143K200.468 14.32 28.70 136.421 20.747 28.30 L144R 136.247 10.71 14.59111.482 10.182 11.35 L144T 129.746 14.68 19.05 108.923 11.961 13.03L146P 116.626 1.15 1.34 115.601 3.429 3.96 T147S 142.175 3.93 5.59130.287 2.605 3.39 T150N 140.724 6.27 8.82 116.923 6.725 7.86 T150S107.327 6.40 6.87 142.000 6.087 8.64 E151A 103.310 12.11 12.51 126.04711.783 14.85 E151L 132.125 4.90 6.48 121.830 6.264 7.63 E151S 115.4236.20 7.15 136.397 4.695 6.40 E151T 128.337 0.00 0.00 110.300 0.000 0.00E151V 111.531 7.31 8.15 99.647 7.420 7.39 E151W 158.415 1.15 1.83 94.9190.895 0.85 K152T 149.169 5.57 8.31 136.747 3.558 4.87 K152W 122.313 2.473.02 134.039 2.868 3.84 E158S 133.038 0.00 0.00 102.519 0.000 0.00 K162E67.857 3.51 2.38 41.026 30.000 12.31 L165F 106.283 11.82 12.57 96.66714.286 13.81 V166Q 155.975 13.35 20.82 117.990 10.953 12.92 V166T183.384 12.69 23.26 136.882 13.056 17.87 E167D 136.745 10.01 13.69162.637 3.784 6.15 I169L 140.177 13.19 18.49 122.272 15.528 18.99 K170R160.710 8.24 13.24 97.128 10.075 9.79 G172A 167.554 7.51 12.59 133.7357.207 9.64 K173R 106.771 9.80 10.46 134.300 7.489 10.06 L174G 114.13012.38 14.13 264.368 13.478 35.63 L174N 154.332 13.27 20.48 126.18618.907 23.86 L174T 124.819 13.06 16.30 144.876 6.098 8.83 N178K 166.8715.27 8.80 103.154 8.021 8.27 N178R 199.596 4.08 8.15 144.957 3.943 5.72H193Q 213.585 15.28 32.64 138.113 18.326 25.31 K195T 126.161 22.48 28.36237.097 15.280 36.23 K195N 130.253 22.38 29.15 96.381 25.487 24.57 K196E90.574 36.80 33.33 154.091 23.500 36.21 K196R 106.100 13.22 14.02 95.14217.663 16.81 F204P 83.571 84.62 70.71 82.418 126.000 103.85 N205A139.223 21.34 29.71 102.031 18.735 19.12 N205E 160.930 19.30 31.0693.313 18.503 17.27 N205L 107.472 10.56 11.35 0.000 #DIV/0! 8.55 N205T145.085 10.05 14.58 110.627 13.054 14.44 V206I 189.274 13.17 24.92111.220 15.575 17.32 K209R 119.794 11.90 14.26 79.535 3.947 3.14 D212N112.626 2.66 3.00 132.249 5.352 7.08 D212S 122.899 8.35 10.27 147.9366.841 10.12 D213A 183.830 26.85 49.36 154.770 13.699 21.20 D213M 159.2556.83 10.88 98.365 6.940 6.83 S215H 109.069 10.04 10.95 78.992 5.758 4.55S215M 174.883 4.20 7.35 74.943 8.957 6.71 N219I 254.438 8.84 22.49291.200 11.264 32.80 E220V 131.985 7.43 9.81 113.610 5.909 6.71 T222G153.033 0.61 0.94 105.454 0.793 0.84 T232F 132.839 12.43 16.51 62.88219.590 12.32 Q233G 280.488 0.00 0.00 127.368 0.000 0.00 Q234M 95.60522.31 21.33 80.766 20.283 16.38 S235A 129.818 11.06 14.36 120.916 12.02614.54 V237C 138.042 0.00 0.00 116.384 0.000 0.00 V237H 122.112 12.4315.18 145.253 7.407 10.76 V237T 167.105 21.26 35.53 126.020 21.457 27.04A238E 94.878 8.17 7.76 142.167 6.682 9.50 A238H 59.585 26.09 15.54204.683 8.345 17.08 T240A 141.283 9.14 12.92 144.667 9.063 13.11 T240Q162.763 14.76 24.02 120.980 13.776 16.67 R248A 113.237 1.05 1.19 124.6502.408 3.00 E249V 142.752 15.29 21.83 111.068 16.462 18.28 P257G 125.2200.78 0.98 112.803 0.677 0.76 K260M 116.690 8.58 10.01 97.396 7.273 7.08S261A 57.547 67.52 38.86 86.173 54.021 46.55 S261K 161.931 16.05 26.00116.159 22.820 26.51 S261N 142.901 10.46 14.95 35.529 13.403 4.76 A267T196.154 35.29 69.23 111.579 38.679 43.16 F273H 122.647 6.00 7.35 119.0375.973 7.11 F273Y 119.713 7.78 9.32 102.772 9.634 9.90 Q276H 74.908 8.936.69 106.393 10.065 10.71 Q276M 98.323 5.64 5.55 104.948 0.000 0.00Q276R 121.431 10.93 13.27 150.180 8.778 13.18 Q276S 110.643 9.95 11.01138.696 8.745 12.13 V277A 140.765 6.97 9.82 129.580 5.167 6.70 V277E175.779 3.75 6.60 195.598 5.222 10.21 V277H 129.434 3.16 4.09 137.6847.014 9.66 V277K 375.721 13.21 49.63 373.799 12.029 44.96 V277M 137.13815.05 20.64 112.084 14.851 16.65 V277N 89.645 7.29 6.54 273.386 6.76218.49 V277Q 119.930 5.70 6.83 116.151 7.772 9.03 V277R 96.071 15.5714.96 171.465 9.801 16.81 V277S 66.260 7.65 5.07 144.916 4.731 6.86V277T 101.010 7.99 8.07 143.311 7.788 11.16 L278E 75.408 5.11 3.85100.179 7.214 7.23 L278G 122.274 6.50 7.94 104.077 7.887 8.21 K279H138.964 14.99 20.83 123.183 20.090 24.75 V287T 145.345 16.49 23.97124.738 12.019 14.99 T289S 104.598 0.98 1.02 98.234 0.699 0.69 G291S184.581 12.17 22.47 119.565 4.156 4.97 G291V 112.807 19.87 22.42 151.03912.609 19.05 E292C 127.307 8.07 10.27 101.126 8.905 9.01 E292F 137.9306.17 8.52 132.340 5.840 7.73 E292H 170.153 8.73 14.85 115.501 11.77513.60 E292R 112.278 12.61 14.16 129.890 11.983 15.56 E292V 163.075 13.2821.66 133.274 11.847 15.79 T293A 128.197 3.38 4.33 57.524 4.248 2.44A298G 212.422 8.77 18.63 86.131 9.322 8.03 L307G 117.857 0.00 0.0091.528 0.000 0.00 S308D 127.652 4.15 5.30 105.846 2.907 3.08 S308K126.882 1.33 1.69 99.169 0.000 0.00 S308N 170.413 5.67 9.66 139.0835.907 8.22 I309E 123.847 16.25 20.12 129.940 14.414 18.73 I309G 102.6017.37 7.56 114.091 6.458 7.37 I309L 153.681 9.58 14.72 106.948 10.90511.66 I309M 123.425 8.02 9.90 136.797 7.065 9.66 I309N 111.901 6.98 7.8197.361 6.470 6.30 I309S 169.951 4.11 6.98 551.493 0.862 4.75 I309T97.936 7.63 7.47 148.864 5.542 8.25 I309V 113.138 1.50 1.70 138.3133.470 4.80 M310G 167.656 11.44 19.18 110.739 12.916 14.30 M310Q 107.23727.81 29.82 106.323 28.254 30.04 M313G 138.095 9.77 13.49 109.231 10.14111.08 M313H 271.914 3.71 10.09 197.024 3.886 7.66 M313K 118.882 0.861.02 107.111 0.555 0.59 M313P 103.654 4.98 5.16 132.802 4.516 6.00 M313R157.272 4.62 7.27 32.845 8.296 2.72 M313T 162.074 7.04 11.40 99.8447.007 7.00 M313Y 120.038 7.52 9.03 103.011 6.846 7.05 K314S 141.924 9.6713.73 132.112 9.066 11.98 K314Y 243.011 5.75 13.98 138.931 10.989 15.27S315A 91.372 16.51 15.08 137.153 9.973 13.68 S315H 151.244 12.06 18.24105.305 10.867 11.44 S315Y 170.968 30.61 52.33 57.827 39.503 22.84 L317A123.510 6.97 8.60 132.724 8.395 11.14 L317I 187.477 12.72 23.84 110.69610.670 11.81 L317K 96.199 3.45 3.31 134.204 3.534 4.74 L317N 127.38212.02 15.31 121.233 14.528 17.61 L317R 238.501 3.87 9.22 99.467 5.6735.64 L317S 90.929 15.54 14.13 85.810 6.423 5.51 L317T 145.964 6.96 10.16154.334 1.087 1.68 L317W 163.704 11.92 19.51 147.606 10.270 15.16 L318D105.543 17.43 18.40 97.970 16.684 16.35 L318H 99.907 4.29 4.29 124.6907.363 9.18 L318R 160.469 5.63 9.03 120.872 6.210 7.51 N321R 164.842 9.5315.71 112.180 8.613 9.66 N321S 102.489 8.29 8.49 108.732 4.534 4.93E324N 104.618 7.72 8.08 131.265 9.124 11.98 T325E 124.837 14.44 18.02106.457 10.577 11.26 N328G 197.098 4.15 8.18 109.722 7.233 7.94 N328Y180.981 10.30 18.64 100.000 10.500 10.50 T335S 107.956 11.57 12.49125.286 6.288 7.88 Q347A 101.395 10.89 11.04 96.213 11.001 10.58 Q347G222.459 8.37 18.63 207.054 9.013 18.66 Q349M 99.531 11.98 11.92 108.04212.330 13.32 Q349R 147.007 11.76 17.29 104.545 13.211 13.81 V351S130.819 0.00 0.00 100.857 0.000 0.00 I353V 132.334 10.45 13.83 138.02511.902 16.43 N356H 100.000 8.54 8.54 130.377 3.912 5.10 N356S 51.9080.00 0.00 125.692 2.516 3.16 S359E 135.589 10.77 14.60 135.104 9.35412.64 S359H 110.422 0.00 0.00 100.809 0.000 0.00 P367A 167.030 12.9421.62 127.366 13.153 16.75 P367G 115.683 0.00 0.00 122.642 0.000 0.00P367K 125.884 5.06 6.36 66.884 10.136 6.78 P367S 74.263 14.39 10.6988.355 16.433 14.52 D368A 121.623 1.45 1.76 81.646 2.111 1.72 D368E166.628 9.18 15.30 97.937 11.462 11.23 D368L 108.977 0.00 0.00 109.3640.969 1.06 D368M 119.744 2.72 3.25 103.662 2.536 2.63 D368R 164.73510.16 16.74 118.140 11.805 13.95 D368T 107.122 2.87 3.07 126.693 3.3664.26 N369R 161.693 6.39 10.34 74.366 6.182 4.60 A371F 180.217 6.19 11.1676.436 5.578 4.26 A371H 957.055 1.81 17.32 89.541 1.697 1.52 A371H111.143 0.00 0.00 95.589 8.610 8.23 A371K 136.514 12.84 17.53 114.35412.454 14.24 A371L 695.108 1.51 10.52 107.003 2.215 2.37 A371L 104.3270.00 0.00 60.232 1.205 0.73 A371R #VALUE! #VALUE! 11.03 #VALUE! #VALUE!14.06 A371R 121.162 0.00 0.00 97.970 2.587 2.53 A371S 147.672 8.38 12.38131.555 16.938 22.28 L374P 392.038 5.77 22.63 123.033 7.365 9.06 E375A88.836 0.00 0.00 134.714 2.050 2.76 E375G 126.880 10.32 13.10 139.03014.673 20.40 E375R 163.180 13.15 21.45 116.431 19.727 22.97 K376D113.100 12.36 13.97 165.064 5.049 8.33 K376E 100.000 13.55 13.55 153.01610.394 15.90 K376Q 125.172 12.75 15.96 90.000 12.057 10.85 K376R 81.68731.63 25.84 199.112 10.372 20.65 K376T 121.133 14.91 18.06 113.387 5.6396.39 K376V 124.221 3.19 3.96 118.583 2.547 3.02 K376Y 102.812 9.24 9.5096.139 12.985 12.48 G377D 110.871 15.72 17.43 132.357 10.550 13.96 G377E130.445 8.04 10.49 128.402 7.401 9.50 G377H 146.855 8.34 12.25 104.83710.117 10.61 G377K 185.922 4.42 8.21 119.751 4.989 5.97 G377R 119.7085.87 7.03 94.749 7.137 6.76 G377S 108.609 6.91 7.51 101.106 7.877 7.96G377T 112.557 17.14 19.29 109.036 18.279 19.93 F380W 147.077 9.97 14.67104.881 9.253 9.70 T381S 135.827 13.41 18.21 112.559 10.315 11.61 R383I527.820 6.33 33.44 98.328 7.522 7.40 R383S 132.894 10.50 13.96 119.46610.545 12.60 K385A 126.096 4.64 5.85 112.706 0.000 0.00 K385Q 137.6299.03 12.43 124.892 7.512 9.38 K385V 112.581 5.12 5.76 80.571 2.979 2.40E389A 306.767 2.13 6.53 224.872 1.824 4.10 E389G 113.253 2.13 2.41139.901 0.000 0.00 E389L 143.219 14.24 20.40 112.185 12.609 14.15 E389Q135.807 11.88 16.14 99.738 12.767 12.73 E389S 165.620 0.00 0.00 93.0300.285 0.27 E392A 112.465 7.27 8.18 155.693 6.376 9.93 E392F 115.619 3.904.51 143.781 3.905 5.61 E392Q 112.993 10.53 11.89 93.789 16.705 15.67E392R 129.528 3.69 4.79 123.407 2.947 3.64 E392V 124.365 7.73 9.61154.768 6.404 9.91 Q393F 139.966 10.59 14.82 101.647 10.171 10.34 Q393M139.696 1.60 2.24 86.966 3.086 2.68 S395A 208.246 12.98 27.04 112.71412.395 13.97 S395H 159.975 12.55 20.07 113.401 10.452 11.85 E396A131.894 8.42 11.10 128.716 9.777 12.58 E396H 210.364 9.19 19.33 128.5713.216 4.14 E396Q 122.977 10.06 12.37 95.938 10.263 9.85 E396S 156.2672.77 4.33 111.753 2.022 2.26 Y399T 130.536 0.00 0.00 122.738 0.050 0.06Y399V 110.592 15.98 17.68 116.018 17.801 20.65 Y399W 122.500 13.76 16.86103.346 11.973 12.37 S401A 122.003 13.90 16.96 99.275 13.024 12.93 S401E125.223 16.30 20.42 128.670 15.000 19.30 S404A 149.379 0.00 0.00 105.4431.102 1.16 L406F 122.805 0.00 0.00 146.122 0.000 0.00 L406N 152.836 6.369.72 131.321 6.705 8.81 S407A 141.351 11.33 16.02 110.376 16.836 18.58S407D 241.053 11.29 27.22 98.135 10.120 9.93 S407P 143.308 6.85 9.81121.898 11.088 13.52 A412Q 146.177 9.54 13.94 99.452 8.511 8.46 A412R140.070 8.92 12.49 123.675 9.390 11.61 A412V 146.804 4.99 7.32 101.7395.383 5.48 D416L 120.820 17.64 21.31 127.662 15.340 19.58 D418R 117.7497.59 8.94 112.193 10.721 12.03 A419H 241.224 8.82 21.27 188.179 5.99911.29 A419K 191.165 10.42 19.91 2022.616 1.523 30.81 D421A 102.111 12.4912.75 301.584 4.510 13.60 D421H 333.471 10.18 33.95 67.652 86.552 58.55D421K 124.190 7.62 9.46 102.316 13.562 13.88 D421N 110.806 14.96 16.58100.116 16.449 16.47 D421Q 104.370 10.72 11.18 143.630 12.400 17.81D421R 138.783 8.85 12.28 137.964 9.778 13.49 D421S 142.171 11.00 15.64166.162 8.564 14.23 A425G 74.810 10.61 7.94 120.947 11.137 13.47 G427Q133.135 2.31 3.08 98.243 8.618 8.47 G427T 125.113 4.81 6.02 119.0583.956 4.71 V428L 137.044 1.81 2.48 109.390 0.990 1.08 D431E 70.178 26.3218.47 95.135 20.739 19.73 D431H 186.490 7.32 13.65 95.071 9.941 9.45D431K 240.835 11.61 27.97 68.277 20.207 13.80 D431L 129.149 10.49 13.54119.177 11.740 13.99 D431N 138.404 9.79 13.55 125.433 9.246 11.60 D431Q232.960 10.83 25.23 109.483 10.716 11.73 D431S 78.069 7.52 5.87 88.7969.135 8.11 F433A 147.286 9.78 14.40 99.486 12.798 12.73 F433H 140.19613.48 18.90 87.943 16.888 14.85 F433I 108.569 11.30 12.27 86.984 16.61614.45 F433K 91.159 11.12 10.14 342.290 3.608 12.35 F433R 128.958 10.5313.58 133.353 9.565 12.75 F433T 161.799 13.66 22.10 134.977 19.229 25.96F433V 1412.071 1.61 22.69 112.033 17.307 19.39 F433W 149.049 10.46 15.59113.585 7.530 8.55 P437I 148.880 2.39 3.56 107.028 1.782 1.91 M438A106.463 10.07 10.72 135.705 10.194 13.83 M438D 105.370 10.16 10.71113.283 2.590 2.93 M438E 115.061 8.00 9.21 113.782 9.120 10.38 M438L65.794 10.06 6.62 214.958 6.526 14.03 M438N 130.428 8.06 10.52 100.66911.889 11.97 M438T 104.058 13.39 13.93 103.691 12.160 12.61 E439A137.279 11.63 15.97 95.555 14.073 13.45 E439A 154.140 4.72 7.28 147.2957.415 10.92 E439C 193.243 14.69 28.38 111.719 15.734 17.58 E439K 124.46413.28 16.52 104.762 10.552 11.05 E439P 118.340 15.59 18.44 87.446 14.99813.12 E439Q 101.589 10.67 10.84 127.358 10.648 13.56 E439T 110.891 14.3615.93 122.975 11.322 13.92 T440D 118.877 11.52 13.69 79.518 18.426 14.65T440H 142.296 4.46 6.34 130.928 7.553 9.89 T440M 84.722 8.83 7.48 86.92912.774 11.10 T440P 111.931 13.54 15.16 91.205 17.272 15.75 T440S 100.43611.17 11.22 131.174 9.810 12.87 E441F 129.315 11.25 14.55 110.874 11.41012.65 E442G 111.216 10.24 11.39 100.210 10.965 10.99 P443E 94.377 5.144.85 130.704 6.789 8.87 P443F 146.612 11.22 16.45 97.932 12.322 12.07P443G 239.171 8.56 20.48 157.960 16.385 25.88 Q444E 81.997 8.54 7.01160.917 9.561 15.38 Q444H 150.301 8.46 12.71 119.665 10.892 13.03 Q444V129.822 13.49 17.51 122.591 10.995 13.48 I445M 85.090 17.25 14.68101.149 15.393 15.57 I445N 106.430 13.89 14.79 87.351 12.945 11.31 I445W117.213 11.70 13.72 100.037 10.983 10.99 Y447E 99.579 16.55 16.48108.969 12.849 14.00 Y447G 143.704 13.77 19.79 103.624 11.563 11.98Y447P 139.152 13.78 19.17 107.737 12.282 13.23 positive 94.998 5.23 4.9796.871 8.456 8.19 control 105.798 4.48 4.74 108.066 5.246 5.67 (OHO)100.000 3.33 3.33 82.7780 3.759 4.59 94.762 19.07 18.07 109.539 16.52918.11 142.024 4.48 6.36 130.947 5.595 7.33 45.115 20.77 9.37 68.01711.035 7.51 53.324 21.95 11.71 74.253 9.960 7.40 59.581 25.24 15.0475.872 16.231 12.31 91.844 19.05 17.50 80.371 13.977 11.23 93.828 13.4712.63 96.630 19.454 18.80 57.773 17.04 9.85 83.536 17.573 14.68 100.00018.56 18.56 148.226 16.239 24.07 74.325 18.29 13.60 61.119 9.286 5.6898.132 8.48 8.32 87.677 10.006 8.77 93.817 9.62 9.02 102.223 9.745 9.9696.922 8.56 8.30 87.993 9.064 7.98 96.648 9.91 9.58 86.891 9.938 8.63n/a (not available; e.g., beyond detection limit)

2. Summary of Results for F204P

For mutant F204P, the results above of tested supernatant from transienttransfection of CHO-S cells incubated in the presence of m-cresol in abHA enzymatic activity assay showed that the F204P mutant protein washighly resistant to 0.4% m-cresol treatment. The results showed that theactivity that remained after 4 hours incubation with 0.4% m-cresol at37° C. was approximately equal to the activity observed when the enzymewas incubated at either 4° C. or at 37° C. in the absence of m-cresol.The positive control (WT PH20-OHO) showed a reduction in activity of 75%and 83% on the day of the assay (as assayed from two different OHOtransfections). This demonstrated that the F204P phenophile was able toretain 60% to 90% or greater of its activity above the residual activityof the wildtype PH20 control enzyme.

In order to confirm the stability of F204P upon m-cresol treatment orexposure to increased temperature, a second transfection of F204P wasperformed in duplicate using CHO-S cells, and clarified supernatant wasagain tested for its stability at 4° C., at 37° C. for 4 hours with 0.4%m-cresol and at 37° C. for 4 hours without 0.4% m-cresol. The resultsconfirmed that the F204P mutant enzyme retained a high amount ofhyaluronidase activity after the 4 hour incubation in m-cresol at 37° C.The results were similar to the results seen in the first screening ofthe mutant, with F204P retaining anywhere from 57% to greater than 90%of its activity above the residual activity of the wildtype PH20 controlenzyme after the 4 hour incubation.

A summary of the enzyme activity of F204P compared to the wildtypecontrol is set forth in Table 13.

TABLE 13 Summary of Enzyme Activity Remaining Activity RemainingActivity after 4 h incubation after 4 h incubation (37° C. + m-cre/ (37°C. + m-cre/37° C.) Net % Increase 4° C.) Net % Increase WT in ActivityOver WT in Activity Over Transfection # F204P (OHO) WT (37° C.) F204P(OHO) WT (4° C.) 1 73.6% 16.4% 57.2% 86.0% 25.3% 60.7% 2 122.3% 25.2%97.1% 109.7% 16.6% 93.1%

Example 6 Large Scale Expression and Purification of pH20 Hit Variant 1.Expression and Purification

HZ24-PH20-IRES-SEAP plasmid DNA containing cDNA encoding one of thevariant PH20 was transfected into monolayer CHO-S cells as generallydescribed in Example 2. CHO-S cells were cultured in shaker flasks usingCD-CHO media supplemented with GlutaMAX (8 mM). On the day oftransfection, 15 flasks were prepared of approximately 300 mL volumecontaining the CHO-S cells at an approximate density of 1.0×10⁶cells/mL. Each 300 mL flask was transfected using 375 μg of plasmid DNAencoding the F204P mutant combined with 375 μL of Freestyle MAXtransfection reagent. The transfected plasmid DNA had a sequence ofnucleotides set forth in SEQ ID NO:4 containing a codon change of TTC toCCT at nucleotide positions 1733-1735, thereby encoding the F204Pmutant. The transfected cells were then allowed to remain in culture for96 hours, whereupon the cells and media were harvested and pooled. Thecells were pelleted by centrifugation (4000×g, 20′), and the supernatantretained for purification of the F204P protein (approximately 4.5liters).

The crude supernatant was concentrated 10× using a 30 kDa Tangentialflow filter (TFF) system (Millipore Pellicon XL, Bimax 30, 200 mL voidvolume; 50 cm² filter surface area) until the volume was approximately450 mL. The permeate was saved for assay to detect flow through of theF204P protein. A free-flow buffer exchange for the retentate was thenperformed using 4 liters of buffer (10 mM NaPO₄; 25 mM NaCl, pH 7.2).The volume of the retentate was reduced again to approximately 200 mL,and then the remaining permeate in the system was purged (void volume˜200 mL) and the system was flushed using approximately 50 mL of bufferto yield a final concentrated product of approximately 450 mL.

An anti-rHuPH20 affinity column was prepared by coupling antigenaffinity purified Rabbit anti-rHuPH20 IgG to CNBr-activated Sepharose 4Fast Flow (GEHealth catalog No. 17-0981-01). Briefly, 0.7 g ofpre-activated Sepharose 4 powder was suspended in 1 mM HCl in a 10 mLglass column for 30 minutes to allow the powder to swell. The solutionwas drained from the column and washed with 15 gel volumes (about 30 mL)of cold 1 mM HCl by gravity. The column was washed with 5 gel volumensof coupling buffer (0.1M NaHCO₃, 0.5M NaCl at pH 8.3). Next, 5 mg ofRabbit anti-rHuPH20 IgG at >1.0 mg/mL in coupling buffer was added tothe column at a protein/gel ratio of 2-3 mg/mL gel. The column wasrotated head to head at 4° C. overnight. The flow-through was collectedfor coupling efficiency determination. The gel was washed with 2 gelvolumes of coupling buffer, and then washed and resuspended in 1 Methanolaminine pH 9.5 for 2 hours at room temperature to block unusedactivated sites. The gel was washed 6 times with 5 gel volumes per washalternating coupling buffer and 0.1 NaAc, 0.5M NaCl, pH 4.5. The gel wasthen washed with 10 gel volumes of TBS (20 mM Tris-HCl, 0.15 M NaCl, pH7.5). The coupling efficiency was determined (1-post-coupling proteinconcentration/pre-coupling protein concentration ×100%). The antibodycoupled gel was stored in TBS with 0.02% NaN₃ at 4° C.

The concentrated supernatant product was subsequently loaded onto aanti-rHuPH20 affinity column at an approximate rate of 5 mL/min. Theelution was performed according to standard procedure using a GE™ AKTAFPLC purification system (GE Healthcare, Product No. 18-1900-26),whereby the protein was eluted via a low pH glycine wash (0.1 Mglycine-HCl, pH 2.5) in 1 mL fractions. Each fraction was immediatelyneutralized by the addition of 100 μL of 1M Tris, pH 7.5.

The eluted protein was assayed by resolving protein bands on a 4-20%SDS-PAGE gradient Tris-glycine gel. SeeBlue®Plus2 Pre-stained MWstandards (Life Teechnologies; Catalog No. LC5925) were used asmolecular weight standards, and 50 ng rHuPH20 (as described inExample 1) was used as a positive control. The polyacrylamide gel wasstained with Instant Blue to show total protein from each fraction. Toconfirm the bands on the gel are PH20, the gel was transferred to a PVDFmembrane (Invitrogen), which was subjected to Western Blot using aRabbit anti-PH20 primary antibody generated by immunizing rabbits withrHuPH20 and an HRP-Goat anti-rabbit secondary antibody (Calbiochem, Cat.No. DC03L).

Then, the flow-through from the initial loading of the affinity columnwas re-loaded onto the column twice due to the low capacity of theaffinity column. All fractions containing the protein were then combinedresulting in a total volume that was approximately 13 mL. This productwas then dialyzed overnight versus four liters of buffer (10 mM NaPO₄,140 mM NaCl, pH 7.2) using a Slide-A-Lyzer Dialysis Cassette G2 (20,000MWCO) with a 15 mL capacity. The buffer was then changed and the productdialyzed against a second fresh four liters of the same buffer. TheF204P protein was then concentrated using an Amicon Ultra Centrifugationcolumn (Millipore; 10,000 MWCO) to a final volume of approximately 450μL (10 minutes at 4000×g).

2. Characterization of Protein

The purified protein was characterized for its protein concentration,activity, and purity.

To determine the protein concentration of the purified protein, aquantification ELISA was performed as described in Example 7. Also,hyaluronidase activity was determined as described in Example 3. Theprotein concentration after centrifugation was estimated to beapproximately 400 μg/mL. The purified protein also was resolved on a4-20% SDS-PAGE gradient Tris-glycine gel, which was then stained withInstant Blue. The staining results demonstrated that the protein wasessentially a single molecular weight protein of approximately 63 kDa,similar to the rHuPH20 control. No appreciable degradative products weredetected by this method. Approximate yields of the protein at varioustimepoints and activity during the purification are described in Table14.

TABLE 14 Characterization of Purification Steps Quant ELISA ActivityAssay Assay Total Protein Total Specific Purification Volume ActivityActivity Conc. Protein Activity Step (mL) (U/mL) (U) (μg/mL) (μg) (U/μg)Supernatant 4500 2.66 11,700 0.046 207 56.5 Conc. after 450 42 18,9000.4 178 105.9 TFF & Buffer Exchange Pooled 0.45 11,741 5283 396 180 35.3Fractions 5-7 after AC, Dialysis & Conc.—A280

The purity of the purified protein was determined by Reverse Phase HPLC(RP-HPLC). The elution time from the reverse phase column wasessentially identical as that observed with the recombinant humanhyaluronidase (HUB), and provides a basis for crude estimation of thepurity of the sample at approximately 80-90%.

Example 7 Quantification Using ELISA

The quantification of PH20 or variants were performed using an ELISAthat captures the protein using a monoclonal anti-rHuPH20 captureantibody. Specifically, one day prior to performing the ELISA, 96-well4HBX plates were coated with capture antibody (Protein G purified rabbitpolyclonal anti-PH20 antibody generated by immunizing rabbits withrHuPH20; 1 mg/mL stock) at 1 μg/mL in 100 mM phosphate (pH 7.2) in atotal volume of 100 μL per well. The plates were stored at 4° C.overnight. On the next day, the plates were washed 5× with 1×PBS at 300μL/well with a plate washer. After each wash, the plated were patted dryon paper towels. Then, the plates were blocked with 200 μL PBScontaining Tween 20 (1×PBST) per well at room temperature for 1 hour.

The standards and samples were added to the plate. For generation of thestandard, a 1 mg/mL stock of rHuPH20 (Example 1) was freshly diluted to50 μg/mL in HEPES pH 7.4 assay buffer as an intermediate stock. Then,for the standards, the 50 μg/mL stock was diluted in duplicates into 360μL of 0.5×PBST at 300 ng/mL for the first standard (first row). For theother standard rows, 240 μL 0.5×PBST were added to each well, and 1:3serial dilutions made. For the transfected supernatant samples, 360 μLper well was added in duplicate into the first row, and each were alsoserially diluted as described above into 0.5× PBST. For purifiedsamples, 100 μL was added per well. The plates were incubated for 2hours at room temperature. After incubation, the plates were washed 5×with 1×PBST at 300 μL/well using a plate washer. After each wash, theplates were patted dry on paper towels.

An HRP-conjugated anti-PH20 antibody was prepared for detection using anHRP conjugation kit (Pierce, Thermo-Fisher; Catalog No. 31489). 1 mg ofa Protein G purified rabbit polyclonal antibody generated by immunizingrabbits with rHuPH20 was diluted in 1 mL PBS and 1 mL of 2× carbonatekit buffer. Next, 100 μL of peroxidase were added to 1 mL of the aboveantibody solution and incubated at room temperature for 1 hour. Then, 10μL NaBH4 stock was added in a fume hood, and the sample incubated atroom temperature for 20 minutes. To quench the reaction, 20 μL ofethanolamine was added and incubated at room temperature for 15 minutes.To this, 1/25 volume 5% human serum albumin (0.1 mL syringe) was addedto give a 2 mg/mL albumin stock reaction. The pH was adjusted to about7.9 by addition of 250 μL of 1 M Tris pH 7.4. The concentration of thestock was 400 μg/mL. The stock solution was further diluted 1/10 in PBSTween20 (0.05%) containing 0.5% human serum albumin and preservatives,and then was sterile filtered. The stock was stored at 4° C. or wasfrozen at −20° C.

Antibodies were detecting using the HRP-conjugated anti-PH20 antibodythat was diluted 1000× into 0.5× PBST. 100 μL of the diluted antibodywas added to all wells of the plate and the plate incubated for afurther 2 hours at room temperature. After incubation, the plates werewashed 5× with 1×PBST at 300 μL/well using a plate washer. After eachwash, the plates were patted dry on paper towels. Then, 100 μL of TMBsubstrate were added to each well and the reaction was stopped after5-10 minutes by adding 100 μL of stop solution per well. The plate wasread at OD₄₅₀.

Example 8 Determination of Enzymatic Activity of pH20

Enzymatic activity of PH20 in samples such as cell cultures,purification fractions and purified solutions was determined using aturbidimetric assay, which is based on the formation of an insolubleprecipitate when hyaluronic acid binds with cetylpyridinium chloride(CPC). The activity is measured by incubating PH20 with hyaluronan for aset period of time (30 minutes) and then precipitating the undigestedhyaluronan with the addition of CDC. The turbidity of the resultingsample is measured at 640 nm. The decrease in turbidity resulting fromenzyme activity on the hyaluronan substrate is a measure of the PH20enzymatic activity. The method is run using a calibration curvegenerated with dilutions of a PH20 assay working reference standard(rHuPH20 standard generated as described in Example 1), and sampleactivity measurements are made relative to this calibration curve.

Dilutions of the sample and standards were prepared in Enzyme DiluentSolution (70 mM NaCl, 0.1% human serum albumin [HSA], 0.67 g/L gelatinhydrolysate in 25 mM PIPES buffer, pH 5.5). The samples were diluted toan appropriate concentration. Hyaluronic acid (HA, average MW of 20-50kDa) from Lifecore Biomedical (Chaska, Minn.) also was prepared at 1mg/mL in substrate solution that contains 25 mM PIPES, 70 mM NaCl at pH5.5. Equal amounts of the above two solutions were mixed to prepare a 1mL reaction mixture and incubated at 37° C. for 30 min. The reaction wasstopped by addition of 4 mL of Cetylpyridinium Chloride Solution (CPC,5.0 mg/mL). After brief vortexing, the turbidity of the sample mixturewas read at 640 nm and the activity was determined by fitting against astandard curve. Specific activity (Units/mg) was calculated by dividingthe enzyme activity (U/mL) by the protein concentration (mg/mL).

Example 9 Stability of F204P-pH20 Variant in Preservative

To confirm the screening results, an amount estimated to be about 450U/mL of the purified F204P protein as described in Example 6 wasformulated in 10 mM sodium phosphate, pH 6.5, 120 mM NaCl, 10 mMmethionine, 0.01% Pluronic F-68, 0.1% phenol and 0.15% m-cresol. A testarticle that also contained an amount estimated to be about 450 U/mLwild type rHuPH20 (generated as described in Example 1) in the sameformulation was also prepared to serve as a control. Each formulationsolution was aliquotted in 0.5 mL and filled into 2 mL USP Type Iborosilicate glass with a chlorobutyl rubber stopper and an aluminumseal. The vials were incubated at 5° C., 30° C. or 37° C. Samples werewithdrawn from the incubator at various times and enzymatic activity wasmeasured as described in Example 8.

The results of the enzymatic activity measurements are shown in Table15. As can be seen, the rHuPH20 wild type control showed a rapiddecrease in activity when incubated at 37° C. in the presence ofphenolic preservatives. In contrast, the F204P mutant showed nosignificant loss in activity throughout the study. The results also showthat activity of PH20 is retained after incubation for up to 4 weeks at5° C. and 30° C. compared to the activity of the rHuPH20 wildtypecontrol not containing the mutation. These results confirm that F204Ptolerates EPB level of preservative (0.1% phenol and 0.15% m-cresol) andis stable at 37° C. for at least up to 6 days at at 5° C. and 30° C. forgreater than one month.

TABLE 15 Stability of rHuPH20 wildtype and F204P mutant incubated atwith preservative PH20 relative PH20 relative PH20 relative activity (%)at activity (%) at activity 5° C. 30° C. (%) at 37° C. ID T0 2 w 4 w 6 d2 w 4 w 2 d 4 d 6 d F204P 100 — 91.8 84.1 100 96.6 105 91.1 95.9wildtype 100 — 81.9 66.7 61.7 60.5 48.6 29.6 15.2 control

Example 10 Stability of F204P-pH20 Variant in Insulin Coformulation

The PH20 variant F204P was tested for its stability in a coformulationcontaining an insulin analog (insulin aspart or insulin lispro).

In the tested coformulations, the insulin lispro was a commercialproduct (Insulin Lispro: Eli Lilly Humalog® (insulin Lispro) 100 U/mL,Lot A572364).

In the tested coformulations, the insulin aspart analog was areprocessed aspart prepared by pooling 12 vials (10 mL each) of acommercial product (Insulin Aspart: Novo Nordisk, NovoRapid® (insulinAspart), Lot XS60195), which was then concentrated using an AmiconUltracel-10 K column concentrator until the final concentration wasabout 5 times the original concentration. The insulin analog wasprecipitated by addition of 1 M sodium acetate, pH 5.3 and 30 mM zincchloride (ZnCl₂, EMD, Cat No. ZX0065-1) at 1/10 of the protein solutionvolume. The solution was placed on ice for 30 minutes followed bycentrifugation at 5600 rpm for 20 minutes in an Avanti J-E Centrifugewith JS-5.3 swinging bucket rotor (Beckman Coulter). The supernatant wasdecanted and the pellet was resuspended and washed with 20 mM sodiumacetate, 2 mM zinc chloride, pH 5.5 solution. The resuspended solutionwas centrifuged as described above. The washing step was repeated atotal of 5 times. A final wash was performed with 20 mM sodium acetate,pH 5.5 to remove all traces of zinc chloride. The resulting proteinpaste was dissolved with water containing 20 mM HCl. After completedissolution, 250 mM Tris, pH 10.7 was added to a final Trisconcentration of 20 mM. The pH of the resulting solution was adjustedsuch that the insulin analog was formulated as described below and theprotein concentration was adjusted to about 15-20 mg/mL. An insulinanalog prepared in this way typically had a yield of about 90%, with aresidual preservative concentration at less than 100 times the startingmaterial.

Briefly, three β) formulations were generated each containing 600 Units(U) of PH20-F204P or wildtype rHuPH20 (generated as described inExample 1) for a total of 6 formulations as set forth in Table 16:

TABLE 16 Summary of Insulin Formulations Buffer Tonicity API Tris/modifier Anti-Ox Metal Surfactant Preservatives PH20 Analog ID pH NaPO₄HCl NaCl Methionine Glycerin Zn F68 Phenol m-Cresol (U/mL) (mg/mL) F1.Humalog + 7.0-7.8 13.2 mM 173.7 mM 0.242 mM 0.315% 600 3.5 F204P F2.Humalog + 7.0-7.8 13.2 mM 173.7 mM 0.242 mM 0.315% 600 3.5 wt F3.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5 F204P F4.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5 wt F5.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5 F204P F6. Aspart +7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5 wt

Each formulation solution was dispensed in 0.5 mL aliquots into 2 mL USPType I borosilicate glass vials with a chlorobutyl rubber stopper and analuminum seal. The vials were incubated at 5° C., 30° C. and 37° C.Samples were withdrawn from the incubator at scheduled time points forenzymatic activity measurements as described in Example 8.

The results of the enzymatic activity measurements for samples incubatedat 37° C., 30° C. and 5° C. are shown in Tables 17-19, respectively. At37° C., the enzymatic activity of samples containing wildtype rHuPH20(F2, F4 and F6) were almost totally lost within two days of incubation.In contrast, after 6 days incubation at 37° C., formulation F3 and F5,which contains PH20-F204P, lost only about 10% and 30%, respectively.The PH20-F204P formulated in commercial Humalog (F1) lost most of itsactivity within 2 days at 37° C. most likely due to the lack of NaCl inthe formulation.

A similar trend for enzymatic activities of ampoules incubated at 30° C.was noted between the PH20-F204P and rHuPH20. For formulations thatcontain an EPA preservative level, the differences between wild type andF204P were dramatic (Table 17; F1 and F5 vs. F2 and F6). When thepreservative concentration was reduced to an EPB level (F3 and F4), theF204P still outperformed wildtype rHuPH20, although there was slightlyhigher rHuPH20 stability compared to EPA conditions. In both EPA and EPBpreservative levels, PH20-F204P was able to maintain its activity up to14 days at 30° C. when 100 mM of NaCl was included in the formulation.

TABLE 17 Enzymatic activity of rHuPH20 wild type and F204P mutantincubated at 37° C. PH20 activity U/mL, (% of remaining activity) IDInitial Activity 2 d 4 d 6 d 2 w F1. Humalog + F204P 583 (100%)  61(10%) 15 (3%) 10 (2%) — F2. Humalog + wt 439 (100%)  4 (1%) — — — F3.Aspart + F204P 625 (100%) 613 (98%) 496 (79%) 570 (91%) 532 (85%) F4.Aspart + wt 566 (100%)  58 (10%) 24 (4%)  4 (1%) — F5. Aspart + F204P657 (100%) 484 (74%) 462 (70%) 478 (73%) 360 (55%) F6. Aspart + wt 596(100%) −1 (0%) — — —

TABLE 18 Enzymatic activity of rHuPH20 wild type and F204P mutantincubated at 30° C. PH20 activity U/mL, (% of remaining activity)Initial ID Activity 6 d 2 w 4 w F1. Humalog + 583 (100%) 345 (59%) 250(43%)  111 (19%) F204P F2. Humalog + wt 439 (100%)  1 (0%) 16 (4%)  −1F3. Aspart + F204P 625 (100%) 601 (96%) 650 (104%) 579 (93%) F4.Aspart + wt 566 (100%) 428 (76%) 390 (69%)  277 (49%) F5. Aspart + F204P657 (100%) 632 (96%) 655 (100%) 561 (85%) F6. Aspart + wt 596 (100%) 145(24%) 65 (11%)   9 (1.5%)

TABLE 19 Enzymatic Activity at 5° C. PH20 activity (U/mL) at 5° C.Initial ID Activity 2 w 4 w F1. Humalog + F204P 583 544 565 F2.Humalog + wt 439 428 404 F3. Aspart + F204P 625 647 607 F4. Aspart + wt566 580 496 F5. Aspart + F204P 657 695 574 F6. Aspart + wt 596 583 519

Example 11 Stability of V58R-pH20 in Insulin Coformulation

A. Stability of V58R-PH20

The PH20 variant V58R was expressed in CHO-S cells as described inExample 2 or Example 6. The transfected plasmid DNA had a sequence ofnucleotides set forth in SEQ ID NO:4 containing a codon change of GTG toCGG at nucleotide positions 1295-1297, thereby encoding the V58R mutant.The V58R mutant was tested for its stability in a coformulationcontaining insulin aspart (insulin aspart analog prepared as describedin Example 10) and under EPA or EPB preservative levels. Briefly, four(4) formulations were generated each containing 600 Units (U) ofPH20-V58R or wildtype rHuPH20 (generated as described in Example 1) asset forth in Table 20. Formulations F1 and F2 represent the EPBpreservative levels while formulations F3 and F4 represent the EPApreservative levels.

TABLE 20 Summary of Insulin Formulations Tonicity API Buffer modifierAnti-Ox Metal Surfactant Preservatives PH20 Analog ID pH NaPO₄ Tris/HClNaCl Methionine Glycerin Zn F68 Phenol m-Cresol (U/mL) (mg/mL) F1.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5 V58R F2.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5 rHuPH20 wtF3. Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5 V58R F4.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5 rHuPH20 wt

Each formulation solution was dispensed in 0.5 mL aliquots into 2 mL USPType I borosilicate glass vials with a chlorobutyl rubber stopper and analuminum seal. The vials were incubated at 30° C. and 37° C. Sampleswere withdrawn from the incubator at scheduled time points for enzymaticactivity measurements as described in Example 8.

The results of the enzymatic activity measurements for samples incubatedat 37° C. and 30° C. are shown in Table 21 and Table 22. At 37° C., theenzymatic activity of samples containing wildtype rHuPH20 (F2 and F4)were almost totally lost within two days of incubation. In contrast,after 6 days incubation at 37° C., formulations F1 (EPB) and F3 (EPA),containing V58R-PH20, lost only about 25% and 40% activity,respectively. At 30° C., the enzymatic activity of samples containingwildtype rHuPH20 also was dramatically reduced in the presence of EPA orEPB preservatives levels within one month of incubation, although therewas a slightly less dramatic loss in activity in the presence of EPBpreservative levels. In contrast, for V58R-PH20, there was no loss ofenzymatic activity for either tested formulation up to 1 month.

TABLE 21 Enzymatic activity of rHuPH20 wild type and V58R mutantincubated at 37° C. PH20 activity U/mL Initial Formulation Activity 2 d4 d 6 d F1. Aspart + V58R 1350 1099 1094 1006 F2. Aspart + rHuPH20 wt677 53 −3 — F3. Aspart + V58R 1189 793 581 464 F4. Aspart + rHuPH20 wt744 12 −9 —

TABLE 22 Enzymatic activity of rHuPH20 wild type and V58R mutantincubated at 30° C. PH20 activity U/mL Formulation Initial Activity 2weeks 4 weeks F1. Aspart + V58R 1350 1368 1208 F2. Aspart + rHuPH20 wt677 422 256 F3. Aspart +V58R 1189 1228 1171 F4. Aspart + rHuPH20 wt 74421 −5

B. Comparison of Stability of F204P and V58R

The PH20 variant V58R-PH20 was compared to F204P for its stability in acoformulation containing insulin aspart (insulin aspart analog preparedas described in Example 10) and under EPA or EPB preservative levels.Briefly, eight (8) formulations were generated as set forth in Table 23.Formulations F1-F4 represent the EPB preservative levels whileformulations F5-F8 represent the EPA preservative levels. FormulationsF3 and F4 and formulations F7 and F8 were identical and represent thewildtype control formulations formulations used for the EPB or EPAstudies, respectively.

TABLE 23 Summary of Insulin Formulations Buffer Tonicity PreservativesAPI Tris/ modifier Anti-Ox Metal Surfactant m- PH20 Analog ID pH NaPO₄HCl NaCl Methionine Glycerin Zn F68 Phenol Cresol U/mL mg/mL F1.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5 V58R F2Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5 F204P F3.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5 rHuPH20wt(1) F4. Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.100% 0.150% 600 3.5rHuPH20 wt(2) F5. Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5V58R F6 Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5 F204P F7.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5 rHuPH20 wt(1) F8.Aspart + 7.3 30 mM 100 mM 5 mM 0.010% 0.315% 600 3.5 rHuPH20 wt(2)

Each formulation solution was dispensed in 0.5 mL aliquots into 2 mL USPType I borosilicate glass vials with a chlorobutyl rubber stopper and analuminum seal. The vials were incubated at 30° C. and 37° C. Sampleswere withdrawn from the incubator at scheduled time points for enzymaticactivity measures as described in Example 8.

The results show that the percentage hyaluronidase activity in thetested formulations after preincubation at 37° C. was slightly greaterfor both PH20 mutants when formulated in EPB and not EPA preservativelevels. While the percent of activity remaining was greater than 80% forboth tested mutants after 6 days incubation in formulations containingEPB preservative levels, it was less in the presence of EPA preservativelevels. For example, the activity remaining at 6 days in EPApreservative levels was slightly less than 80% after 6 days forF204P-PH20, while it was only about 40% for V58R-PH20. Hence, theresults also show that at 37° C., V58R-PH20 is somewhat less stable thanthe F204P-PH20, in particular in a formulation with EPA preservativelevels. After incubation at 30° C. for at least a week, the F204P-PH20and V58R-PH20 were stable and exhibited almost 100% initial activity inthe presence of both EPA and EPB preservative levels. In contrast,rHuPH20 exhibited only about 40% of its initial activity after 4 weeksat 30° C. in the presence of EPB preservative levels, while it exhibitedno detectable activity after 4 weeks at 30° C. in the presence of EPApreservative levels.

Example 12 Expression of F204P-pH20 Using a Lentivirus Expression Vector

A lentivirus expression vector, pLV-EF1a-PH20(F204P)-IRES-GFP-Bsd wasgenerated containing a codon-optimized mutant hyaluronidase cDNAencoding F204P-PH20. The sequence of pLV-EF 1a-PH20(F204P)-IRES-GFP-Bsdis set forth in SEQ ID NO:925. The pLV-EF1a-PH20(F204P)-IRES-GFP-Bsdvector contains an ampicillin resistance gene (AmpR) located atnucleotides 8611-9471, an EF1a promoter at residues 1933 to 2327, anIRES at residues 4786-5370, a GFP-Bsd at residues 5394-6527 andnucleotides encoding F204P-PH20 at residues 3369-4781.

Lentivirus was produced as described in Bandaranayake et al. ((2011)Nucleic Acids Research, 39:e143). Briefly, 293T cells (ATCC) were platedat 6×10⁶ cells onto 10 cm tissue culture plates. After 24 hours, 6 μg ofpsPAX2 (SEQ ID NO:926; Addgene plasmid No. 12260), 3 μg of PMD2.G (SEQID NO:927; Addgene plasmid #12259) and 9 μg lentiviral vector plasmidpLV-EF1a-PH20(F204P)-IRES-GFP-Bsd were mixed in 1.5 mL Opti-MEM (LifeTechnologies). 45 μL of Lipofectamine 2000 (LF2000; Life Technologies)were diluted into 1.5 mL Opti-MEM (Life Technologies). The DNA andLF2000 were mixed gently, and incubated at room temperature for 20minutes to allow the DNA and lipid to form complexes. In the meantime,the overnight culture medium was replaced with 5.0 mL DMEM+10% FBSwithout antibiotics. A volume of 3.0 mL containing the DNA-LF2000complexes were added to the 293T cells. The medium containing theDNA-LF2000 complexes was replaced with 10 mL complete medium at 12-16hours post-transfection. The supernatant was collected at 48 hourspost-transfection and the medium was transferred to a polypropylenestorage tube. The virus-containing medium was spun at 1300 rpm for 5minutes to pellet any 293T cells that were carried over duringcollection. The supernatant was carefully transferred to a sterilepolypropylene storage tube.

CHO-S cells (Invitrogen) were grown in CHO-S media (Invitrogen) withshaking at 120 rpm at 37° C. and 5% CO₂ in vented 125-mL shake flasks(Nalgene). For transduction, CHO-S cells were added to wells of asix-well plate at 2×10⁶ cells per well in 2 ml of CHO-S media containing4 μg/mL hexadimethrine bromide at a final concentration of 4 μg/mL(Polybrene; SIGMA). Virus was added to each well at a multiplicity ofinfection (MOI) of 10 and the cells were incubated with shaking (120rpm) at 37° C. and 5% CO₂ for 6 hours. The cells were then harvested andpelleted by low speed centrifugation (500×g, 5 min). The transductionmedium was removed and replaced with 10 mL of fresh CHO-S medium(Invitrogen) supplemented with GlutaMax (50 mL/liter) and transferred toa T-25 flask. Three days post infection, blasticidin (Invitrogen) wasadded to the growth medium at a concentration of 1 μg/mL. The medium waschanged regularly at 3-4 day intervals, and the cells were transferredto a T75 flask for expansion. Two weeks after the initial infection, thecells were expanded to shaker flasks and maintained in culture usingmedium containing 1 μg/mL blasticidin. F204P-PH20 protein secreted intothe CHO-S medium was collected and purified by affinity chromatographyusing an anti-rHuPH20 affinity column as described in Example 6. Theprotein was prepared in standard API buffer (10 mM Histidine, 130 mMNaCl, pH 6.5).

Example 13 Analysis of Secondary Structure and Melting Temperature

The secondary structure and melting temperature of the PH20 variantF204P was tested and compared to wild-type rHuPH20 (generated asdescribed in Example 1) to further assess stability of the variant. Thesecondary structure was tested by circular dichroism. A Jasco J-810-1505equipped with PTC-4245 was employed for the CD spectral measurement andthe CD spectra were collected by Spectra Manager (Version 1.5, Jasco).Procedures for instrumental set up and data collection are described inTable 24.

TABLE 24 CD Spectroscopy Operation Conditions Parameters ConditionsNitrogen flow rate 25 W/h Sample temperature 30-75° C. Sampleconcentration Approx. 0.1 mg/mL Cell pathlength 1 mm Wavelength 220 nm Data pitch 1° C. Delay time 60 seconds Temperature slope 1° C./minSensitivity standard Response 4 seconds Band width 1 nm

1. Sample Preparation and Measurement Two hundred (200) μL of a 0.1mg·mL protein sample diluted in Mcllvaine's buffer (McIlvaine (1921) JBC49:183) adjusted to pH 6.5 were prepared. A series of samples of theF204P variant were also generated that varied in pH by adjustment usingMcllvaine's buffer to a pH range from 5.0 to 7.5 as set forth in Table25. In addition, samples also were generated by adjusting the NaClconcentration to 17.5 mM to 140 mM as set forth in Table 26. Sampleswere filtered using a 0.2 μm syringe filter prior to measurement.Similar samples were generated for rHuPH20. Then, 200 μL samples weretransferred to a rectangular cuvetted having a 1 mm width and seated onJasco J-810 spectropolarimeter. CD spectra of the samples were collectedunder the conditions described in Table 20. The melting temperature(T_(m)) was calculated using Spectra Manager (v 1.5, Jasco) from the CDspectral intensity measured at the temperature range from 30° C. to 75°C. The cuvettes were cleaned by Chromerge® cleaner (C577-12, Fisherscientific) between individual sample loading and after the run.

TABLE 25 Sample pH and concentration F204P Target Actual F204P Bufferconcentration pH pH (μL) (μL) (mg/mL) 5.0 4.92 25 175 0.1 5.5 5.38 25175 0.1 6.0 5.99 25 175 0.1 6.5 6.49 25 175 0.1 7.0 7.00 25 175 0.1 7.57.5 25 175 0.1

TABLE 26 Sodium Concentration in Samples at pH 6.5 Target NaCl NaCl,Buffer F204P concentration 2.8M F204P at pH 6.5 concentration (mM) (μL)(μL) (μL) (mg/mL) 17.5 0.00 25 175 0.1 50.0 2.32 25 172.7 0.1 75.0 4.1125 170.9 0.1 100.0 5.89 25 169.1 0.1 140.0 8.75 25 166.3 0.1

2. Results

The results show that the secondary structure of F204P is similar torHuPH20. As a function of temperature, circular dichroism showed that achange in the absorption was measured with increasing temperatures. As afunction of pH, the T_(m) distribution was closely comparable for bothF204P and rHuPH20 and the highest T_(m) for each was obtained between pH5.5 and pH 6.0. The results, however, showed that T_(m) of the F204Pvariant was approximately 9° C. higher at all tested ranges thanwildtype rHuPH20. This result indicated that the F204P mutant is morestable against thermal stress conditions. As a function of salt, theresults show that the F204P and wildtype rHuPH20 both exhibited anincreasing T_(m) with higher salt concentration, showing that both havea proportional inclination toward salt concentration.

Example 14 Assessment of Enzymatic Activity in an Intradermal TrypanBlue Dispersion Assay

Spreading activity of the PH20 variant F204P was assessed using a dyedispersion in vivo assay. Briefly, purified PH20 variant F204P (preparedas described in Example 12) and wild-type rHuPH20 (prepared as describedin Example 1) were both formulated in API buffer (10 mM Histidine, 130mM NaCl, pH 6.5) at a concentration of 10,000 U/mL. The stocks werefurther diluted to three target concentrations of 1000, 100 and 10 U/mLby serial 1:10 dilutions in API buffer. Purified proteins (eitherrHuPH20 or F204P-PH20) were diluted 1:1 with 0.4% Trypan Blue (0.4%liquid solution; Catalog No. 15250, Invitrogen) to give a finalconcentration of 5, 50 and 500 U/mL protein, each containing 0.2% trypanblue. A vehicle control (API buffer) also was prepared. Forty-two (42)female NCr nu/nu homozygous mice were used in the study with six miceused per group as set forth in Table 27.

TABLE 27 Summary of Treatment Groups for Dye Dispersion Study No. FinalDose with Injection of Test Trypan Blue Trypan Volume Group Mice Article(Units/mL) Blue (mL) 1 6 Control 0 0.2% 0.04 2 6 rHuPH20 5 0.2% 0.04 3 6rHuPH20 50 0.2% 0.04 4 6 rHuPH20 500 0.2% 0.04 5 6 F204P-PH20 5 0.2%0.04 6 6 F204P-PH20 50 0.2% 0.04 7 6 F204P-PH20 500 0.2% 0.04

Forty (40) μL of samples were administered by a single intradermalinjection. The area of dye dispersion was measured at 2.5, 5, 10, 15 and20 minutes post-injection and was recorded by photographic imaging byphotograph of the injection site with a Nikon D90 digital camera with 60mm prime micro-lens. A laser distance meter (Leica D3) was used toaccurately position the camera at a pre-determined distance from theTrypan Blue dye area on the animal. The area of the dye was determinedusing Image-Pro Analyzer 7.0 (MediaCybernetics, Inc). The calculatedareas were expressed as mm².

The results are set forth in Table 28. The results showed that thedispersion activity of the PH20 variant F204P was substantiallyidentical to the dispersion activity of rHuPH20. The ability to increasethe area of dye dispersion was dose-dependent, with both proteins havinggreatest activity at 500 U/mL. The results also showed that the area ofdye dispersion increased with time post-intradermal injection. The areasof dye dispersion of rHuPH20 and F204P-PH20 were significantly greaterthan the areas of dye dispersion for the controls (p<0.05) at all timepoints when formulated at all concentrations (5, 50 and 500 U/mL) withthe exception of rHuPH20 at the lowest concentration (5 U/mL). Whencompared to each other, rHuPH20 and F204P-PH20 showed similar dispersioneffects, although there was a significant difference in dispersionbetween the two groups at 5 U/mL and 500 U/mL but not at 50 U/mL. Insum, the results show that both rHuPH20 and F204P-PH20 provided astatistically significant increase in the area of dye dispersioncompared to the vehicle control.

TABLE 28 Trypan Blue Dispersion Group Area (mm2) Avg. (n = 6) 2.5 min 5min 10 min 15 min 20 min 1: Control 37.44 ± 2.81 38.16 ± 3.33 43.71 ±2.12 45.70 ± 2.38 48.77 ± 2.14 2: rHuPH20 (5 U/mL) 36.68 ± 2.83 42.31 ±2.57 45.41 ± 2.75 46.72 ± 3.35 49.61 ± 2.97 3: rHuPH20 (50 U/mL) 39.24 ±1.20 44.90 ± 1.44 46.96 ± 1.70 50.08 ± 2.07 53.50 ± 1.59 4: rHuPH20 (500U/mL) 44.72 ± 1.35 50.21 ± 1.92 57.47 ± 1.29 59.77 ± 1.25 57.17 ± 3.285: F204P (5 U/mL) 39.65 ± 1.53 46.09 ± 2.73 48.07 ± 1.43 52.54 ± 2.0154.11 ± 1.01 6: F204P (50 U/mL) 38.10 ± 1.92 47.07 ± 2.12 51.48 ± 2.1455.24 ± 1.90 58.34 ± 2.89 7: F204P (500 U/mL) 46.58 ± 1.67 54.06 ± 2.5258.96 ± 1.85 64.37 ± 1.72 64.44 ± 2.17

Example 15 Assessment of Enzymatic Activity By Dermal BarrierReconstitution

Activity of F204P-PH20 was assessed and compared to rHuPH20 to measurethe amount of time required for the dermal barrier to reconstituteitself after intradermal hyaluronidase administration. Dermalreconstitution was evaluated by comparing the duration of thehyaluronidase spreading activity as assessed by monitoring the area ofdiffusion of 0.4% Trypan Blue over time. The proteins used in the studywere purified PH20 variant F204P (prepared as described in Example 12)and wild-type rHuPH20 (prepared as described in Example 1) that wereboth formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5).Vehicle (API buffer) was used as a control. Male NCr nu/nu homozygousmice were used in the study with three animals per time point for atotal of fifteen mice used per group as set forth in Table 29.

TABLE 29 Summary of Treatment Groups for Dermal Barrier ReconstitutionStudy No. Time Final Injection of Points Test Dose Volume Group Mice (h)Article (Units/mL) (mL) 1 15 0.5, 1, Control 0 0.04 4, 24, 48 2 15 0.5,1, rHuPH20 100 0.04 4, 24, 48 3 15 0.5, 1, F204P 100 0.04 4, 24, 48

All mice received two intradermal doses of vehicle control or rHuPH20 orF204P-PH20 at 100 U/mL in 0.04 mL at study time 0. The same control ortest article was injected on the opposing lateral sides of each animal(right, R; left, L). Injection sites were marked with a permanentmarker. Trypan Blue Stain (0.4% liquid solution; 15250, Invitrogen) wasadministered at a volume of 0.04 mL by intradermal injection at the sameinjection site at 0.5, 1, 4, 24 and 48 hours post-injection of testarticle or control. At 5 and 20 minutes post-injection of the TrypanBlue Stain, the area of the dye at the injection site was measured bydigital imaging of the region as described in Example 14.

The results are set forth in Table 30. The results show that when thearea of dye dispersion was measured at various time points afteradministration of the test article or control, there was a statisticallysignificant increase in the area of dye dispersion at 30 min and 1 hourpost-injection of rHuPH20 or F204P-PH20. By 4 hours post-administrationof the enzymes, however, there was not a statistically significantincrease in the area of dye dispersion compared to control. In addition,no statistically significant differences in the area of dye dispersionwas observed between the rHuPH20 and F204P-PH20 treatment groups.Therefore, the duration of the spreading activity of rHuPH20 and F204Pwere similar and show that rHuPH20 and F204P-PH20 have comparable invivo performance.

TABLE 30 Dermal Reconstitution min time post- Point injection VehiclerHuPH20 F204P-PH20 30 5 49.96 ± 2.05 80.84 ± 8.03 80.76 ± 4.46 20 64.42± 2.49 94.55 ± 7.09 95.75 ± 5.18  1 hour 5 58.01 ± 3.21 82.56 ± 6.4077.11 ± 3.18 20 65.19 ± 6.21 96.19 ± 6.39 91.45 ± 1.73  4 hour 5 52.10 ±3.47 67.19 ± 2.39 67.33 ± 3.93 20 57.69 ± 3.92 81.15 ± 4.45 82.21 ± 4.1424 hour 5 49.87 ± 3.25 59.01 ± 2.15 54.91 ± 3.54 20 57.15 ± 3.47 67.65 ±2.27 62.91 ± 3.30 48 hour 5 53.64 ± 2.99 53.53 ± 4.88 55.64 ± 7.19 2061.57 ± 4.02 66.33 ± 4.12 63.11 ± 5.97

Example 16 In Vivo Pharmacokinetics of F204P-PH20 Compared to rHuPH20

The pharmacokinetics (PK) of rHuPH20 and F204P-PH20 were comparedfollowing intravenous tail-vein administration by measuring the plasmahyaluronidase levels over time after administration. The proteins usedin the study were purified PH20 variant F204P (prepared as described inExample 12; batch concentration 1.02 mg/mL) and wild-type rHuPH20(prepared as described in Example 1; batch concentration 0.95 mg/mL)formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5). Theproteins were prepared at a concentration of 0.087 mg/mL in API bufferfor a dose volume of about 5 mL. An animal that was not administeredwith protein was used a control (pre-dose control). Forty two (42) maleCD-1 mice (˜20-30 grams) were used in the study with six animals pertreatment group as set forth in Table 31.

TABLE 31 Pharmacokinetics of Single Intravenous Dose of rHuPH20 orF204P-PH20 Dose number of Test Dose Volume Group animals (No.) Article(mg/kg) (mL/kg) Euthanasia 1 6 (Nos. 1-6) no treatment N/A N/A pre-dose2 6 (Nos. 7-12) rHuPH20 0.433 5 1 min 3 6 (Nos. 13-18) rHuPH20 0.433 5 5 ± 1 min 4 6 (Nos. 19-24) rHuPH20 0.433 5 10 ± 2 min 5 6 (Nos. 25-30)F204P-PH20 0.433 5 1 min 6 6 (Nos. 21-36) F204P-PH20 0.433 5  5 ± 1 min7 6 (Nos. 37-42) F204P-PH20 0.433 5 10 ± 2 min

Mice were intravenously administered 0.433 mg/kg rHuPH20 or F204P-PH20by tail vein injection. Blood samples were obtained from animals 1minute, 5 minutes and 10 minutes post-administration. Blood samples wereobtained by terminal bleed (cardiac puncture) and collected into bloodcollection tubes containing the anti-coagulant EDTA for the preparationof plasma. Blood samples were centrifuged at 500 g for 10 minutes andthe plasma removed and frozen at −80° C. until assessment ofhyaluronidase activity using the microturbidity assay described inExample 8.

The results are set forth in Table 32. The results show thathyaluronidase activity is detected in plasma prior to treatment with thehyaluronidase. Within 1 minute post-treatment with either rHuPH20 orF204P-PH20 hyaluronidase, there is a detectably high amount ofhyaluronidase activity present in the plasma, which is similar betweenboth treatment groups. Over time, the hyaluronidase activity rapidlydecreases for both treatment groups, although there is detectablyhyaluronidase activity present in the plasma 10 minutespost-administration. At the 5 minute and 10 minute post-administrationtime points, activity in the plasma in animals treated with F204P-PH20is greater than in animals treated with rHuPH20. This shows thatF204P-PH20 exhibits somewhat greater activity for a prolonged timeperiod, and therefore exhibits greater half-life in vivo than rHuPH20.

TABLE 32 rHuPH20 and F204P-PH20 Activity (U/mL) in Mouse Plasma K2EDTATime Point (min) Predose 1 minute 5 minute 10 minute Animal AnimalAnimal Animal Protein No. U/mL No. U/mL No. U/mL No. U/mL rHuPH20 1 BQL7  235^(a) 13 18.3 19 3.76 2 BQL 8   13.5 14 7.70 20 3.70 3 BQL 9 278 158.85 21 2.64 4 BQL 10 328 16 10.5 22 2.70 5 BQL 11 356 17 12.8 23 2.36 6BQL 12 287 18 18.0 24 2.80 F204P-PH20 1 BQL 25 249 31 48.0 37 11.5 2 BQL26 223 32 21.6 38 11.4 3 BQL 27 246 33 38.4 39 10.1 4 BQL 28 246 34 38.640 12.2 5 BQL 20     0.696 35 38.2 41 10.8 6 BQL 30 257 36 28.5 42 10.2BQL—Below Quantifiable Limit <0.625 U/mL with minimum required dilution^(a)Hemolyzed

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

What is claimed:
 1. A modified PH20 polypeptide, comprising one or moreamino acid modifications in an unmodified PH20 polypeptide, wherein: theunmodified PH20 polypeptide consists of the amino acid sequence selectedfrom among SEQ ID NOs: 3, 7 and 32-66; amino acid modifications areselected from among amino acid replacements(s), deletion(s), and/orinsertion(s); the modified PH20 polypeptide comprises an amino acidreplacement at a position corresponding to residue 317, with referenceto amino acid positions set forth in SEQ ID NO:3; the replacement at theposition corresponding to residue 317 is selected from among A, I, K, M,Q, and R; corresponding amino acid positions are identified by alignmentof the PH20 polypeptide with the polypeptide having the amino acidsequence of SEQ ID NO:3; and the modified PH20 polypeptide has at least91% sequence identity to a polypeptide having the amino acid sequenceselected from among SEQ ID NOs: 3, 7 and 32-66.
 2. The modified PH20polypeptide of claim 1, wherein the modified PH20 polypeptide has atleast 95% sequence identity to the amino acid sequence selected fromamong SEQ ID NOs: 3, 7, and 32-66.
 3. The modified PH20 polypeptide ofclaim 1 that has increased resistance to or stability in denaturingconditions compared to an unmodified PH20 polypeptide that does notcontain the amino acid modification(s).
 4. The modified PH20 polypeptideof claim 1 that exhibits increased hyaluronidase activity compared tothe unmodified PH20 polypeptide not containing the amino acidreplacement at position
 317. 5. The modified PH20 polypeptide of claim 1that is a soluble PH20 polypeptide.
 6. The modified PH20 polypeptide ofclaim 1, wherein the replacement at the position corresponding toresidue 317 is Q.
 7. The modified PH20 polypeptide of claim 1, whereinthe replacement at the position corresponding to residue 317 is K. 8.The modified PH20 polypeptide of claim 1, wherein the unmodified PH20polypeptide comprises the amino acid sequence selected from among SEQ IDNOs: 3 and 32-66.
 9. The modified PH20 polypeptide of claim 1, whereinthe unmodified PH20 polypeptide consists of the amino acid sequenceselected from among SEQ ID NOs: 3 and 32-66.
 10. The modified PH20polypeptide of claim 6, wherein the unmodified PH20 polypeptide consistsof the amino acid sequence selected from among SEQ ID NOs: 3 and 32-66.11. The modified PH20 polypeptide of claim 1, wherein the unmodifiedPH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35.12. The modified PH20 polypeptide of claim 1, wherein the unmodifiedPH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32.13. The modified PH20 polypeptide of claim 6, wherein the unmodifiedPH20 polypeptide consists of the amino acid sequence of SEQ ID NO:35.14. The modified PH20 polypeptide of claim 6, wherein the unmodifiedPH20 polypeptide consists of the amino acid sequence of SEQ ID NO:32.15. The modified PH20 polypeptide of claim 1, comprising the sequence ofamino acids set forth in any of SEQ ID NOs: 604-606, 608, and 609, or asequence of amino acids that exhibits at least 91% sequence identity tothe sequence of amino acids set forth in any of SEQ ID NOs: 604-606,608, and 609, and that contains an amino acid replacement at the residuecorresponding to residue 317 with reference to SEQ ID NO:3.
 16. Themodified PH20 polypeptide of claim 1, comprising the sequence of aminoacids set forth in SEQ ID NO:608 or a sequence of amino acids thatexhibits at least 91% sequence identity to the sequence of amino acidsset forth in SEQ ID NO:608 and that contains the amino acid replacementQ at the residue corresponding to residue 317 with reference to SEQ IDNO:3.
 17. The modified PH20 polypeptide of claim 1 that comprises one ormore modifications of the polypeptide selected from among glycosylation,sialylation, albumination, farnesylation, carboxylation, hydroxylation,and phosphorylation.
 18. The modified PH20 polypeptide of claim 1 thatis glycosylated.
 19. The modified PH20 polypeptide of claim 18, whereinthe polypeptide is a glycoprotein that comprises an N-acetylglucosaminemoiety linked to each of at least three asparagine (N) residues.
 20. Themodified PH20 polypeptide of claim 1 that is conjugated to a polymer.21. The modified PH20 polypeptide of claim 20, wherein the polymer isdextran or polyethylene glycol (PEG).
 22. The modified PH20 polypeptideof claim 1, further comprising a heterologous signal sequence, whereinthe unmodified PH20 polypeptide consists of the amino acid sequenceselected from among SEQ ID NOs: 3 and 32-66.
 23. A chimeric polypeptide,comprising the modified PH20 polypeptide of claim
 1. 24. Apharmaceutical composition, comprising the modified PH20 polypeptide ofclaim
 1. 25. The modified PH20 polypeptide of claim 6, wherein: theunmodified PH20 polypeptide consists of the amino acid sequence of SEQID NO:32; and the amino acid sequence of the modified PH20 polypeptidehas at least 95% sequence identity to the amino acid sequence of SEQ IDNO:32.
 26. The modified PH20 polypeptide of claim 6, wherein: theunmodified PH20 polypeptide consists of the amino acid sequence of SEQID NO:35; and the amino acid sequence of the modified PH20 polypeptidehas at least 95% sequence identity to the amino acid sequence of SEQ IDNO:35.
 27. The pharmaceutical composition of claim 24, furthercomprising a therapeutically active agent formulated in the samecomposition or in a separate composition.
 28. The pharmaceuticalcomposition of claim 27, wherein the therapeutically active agent is apolypeptide, a protein, a nucleic acid, a drug, a small molecule, or anorganic molecule.
 29. The pharmaceutical composition of claim 27,wherein the therapeutically active agent is selected from among achemotherapeutic agent, an analgesic agent, an anti-inflammatory agent,an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent,an anti-Parkinson agent, an anti-malarial agent, an anticonvulsantagent, an anti-depressant agent, an antiarthritics agent, an anti-fungalagent, an antihypertensive agent, an antipyretic agent, an anti-parasiteagent, an antihistamine agent, an alpha-adrenergic agonist agent, analpha blocker agent, an anesthetic agent, a bronchial dilator agent, abiocide agent, a bactericide agent, a bacteriostat agent, a betaadrenergic blocker agent, a calcium channel blocker agent, acardiovascular drug agent, a contraceptive agent, a decongestant agent,a diuretic agent, a depressant agent, a diagnostic agent, an electrolyteagent, a hypnotic agent, a hormone agent, a hyperglycemic agent, amuscle relaxant agent, a muscle contractant agent, an ophthalmic agent,a parasympathomimetic agent, a psychic energizer agent, a sedativeagent, a sympathomimetic agent, a tranquilizer agent, a urinary agent, avaginal agent, a viricide agent, a vitamin agent, a non-steroidalanti-inflammatory agent, an angiotensin converting enzyme inhibitoragent, and a sleep inducer.
 30. The pharmaceutical composition of claim27, wherein the therapeutically active agent is an antibody.
 31. Amethod for treating a hyaluronan-associated disease or condition,comprising administering to a subject a modified PH20 polypeptide ofclaim
 1. 32. The method of claim 31, wherein the hyaluronan-associateddisease or condition is an inflammatory disease or a tumor or cancer.33. The method of claim 32, wherein the hyaluronan-associated disease orcondition is a solid tumor.
 34. The modified PH20 polypeptide of claim 1that is modified by conjugation to a moiety selected from among amultimerization domain, a toxin, a detectable label, and a drug.
 35. Themodified PH20 polypeptide of claim 34, wherein the modified PH20polypeptide is conjugated to a multimerization domain that is an Fcdomain.
 36. The modified PH20 polypeptide of claim 1 that isC-terminally truncated, whereby the polypeptide is soluble.